# Patent

## Title
Methods for diagnosing stomach cancer using MicroRNAs

## Abstract

The present invention provides novel methods and compositions for the diagnosis and treatment of solid cancers. The invention also provides methods of identifying inhibitors of tumorigenesis.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is filed under 35 U.S.C §111(a) as a divisional application which claims priority under 35 U.S.C. §119, 35 U.S.C. §120 and the Patent Cooperation Treaty to: parent application U.S. Ser. No. 12/160,061 filed under 35 U.S.C. §371 on Jul. 3, 2008, now allowed; which claims priority to PCT/US2007/000159 filed under the authority of the Patent Cooperation Treaty on Jan. 3, 2007, published; which claims priority to U.S. Provisional Application Ser. No. 60/756,585 filed under 35 U.S.C. §111(b) on Jan. 5, 2006; the disclosures of all priority applications are incorporated herein by reference.


GOVERNMENT SUPPORT
This invention was supported, in whole or in part, by Program Project Grant Nos. P01CA76259, P01CA81534 and P30CA56036 from the National Cancer Institute. The Government has certain rights in this invention.


BACKGROUND OF THE INVENTION
Cancer, the uncontrolled growth of malignant cells, is a major health problem of the modern medical era and is one of the leading causes of death in developed countries. In the United States, one in four deaths is caused by cancer (Jemal, A. et al., CA Cancer J. Clin. 52:23-47 (2002)). Among cancers, those that arise from organs and solid tissues, known as solid cancers (e.g., colon cancer, lung cancer, breast cancer, stomach cancer, prostate cancer, pancreatic cancer) are among the most-commonly identified human cancers.
For example, prostate cancer is the most frequently diagnosed noncutaneous malignancy among men in industrialized countries, and, in the United States, 1 in 8 men will develop prostate cancer during his life (Simard, J. et al., Endocrinology 143(6):2029-40 (2002)). The incidence of prostate cancer has dramatically increased over the last decades and prostate cancer is now a leading cause of death in the United States and Western Europe (Peschel, R. E. and J. W. Colberg, Lancet 4:233-41 (2003); Nelson, W. G. et al., N. Engl. J. Med. 349(4):366-81 (2003)). An average 40% reduction in life expectancy affects males with prostate cancer. If detected early, prior to metastasis and local spread beyond the capsule, prostate cancer can often times be cured (e.g., using surgery). However, if diagnosed after spread and metastasis from the prostate, prostate cancer is typically a fatal disease with low cure rates. While prostate-specific antigen (PSA)-based screening has aided early diagnosis of prostate cancer, it is neither highly sensitive nor specific (Punglia et al., N. Engl. J. Med. 349(4):335-42 (2003)). This means that a high percentage of false negative and false positive diagnoses are associated with the test. The consequences are both many instances of missed cancers and unnecessary follow-up biopsies for those without cancer.
Breast cancer remains the second leading cause of cancer-related deaths in women, affecting more than 180,000 women in the United States each year. For women in North America, the life-time odds of getting breast cancer are now one in eight. Although the discovery of BRCA1 and BRCA2 were important steps in identifying key genetic factors involved in breast cancer, it has become clear that mutations in BRCA1 and BRCA2 account for only a fraction of inherited susceptibility to breast cancer (Nathanson, K. L., et al., Human Mol. Gen. 10(7):715-720 (2001); Anglican Breast Cancer Study Group. Br. J. Cancer 83(10):1301-08 (2000); and Syrjakoski, K., et al., J. Natl. Cancer Inst. 92:1529-31 (2000)). Despite considerable research into therapies for breast cancer, breast cancer remains difficult to diagnose and treat effectively, and the high mortality observed in breast cancer patients indicates that improvements are needed in the diagnosis, treatment and prevention of the disease.
Excluding skin cancer, colorectal cancer is the third most frequently diagnosed cancer in the United States and Canada (after lung and breast in women, and lung and prostate in men). The American Cancer Society estimates that there will be approximately 145,000 new cases of colorectal cancer diagnosed in the U.S. in 2005 (Cancer Facts and Figures 2005. Atlanta, Ga.: American Cancer Society, 2005. Available at www.cancer.org/docroot/STT/stt—0.asp, accessed Dec. 19, 2005). Colorectal cancer is the second leading cause of cancer death among men and women in the United States and Canada (after lung cancer).
The annual incidence of pancreatic cancer is nearly equivalent to the annual mortality, estimated to be 31,860 and 31,270, respectively, in the U.S. in 2004 (Cancer Facts and Figures 2004. Atlanta, Ga.: American Cancer Society, 2004. Available at www.cancer.org/docroot/STT/stt—0—2004.asp, accessed Aug. 21, 2005). Patients with locally advanced and metastatic pancreatic cancer have poor prognoses, and diagnosis generally occurs too late for surgery or radiotherapy to be curative (Burr, H. A., et al., The Oncologist 10(3): 183-190, (2005)). Chemotherapy can provide relief of symptoms for some patients with advanced pancreatic cancer, but its impact on survival has been modest to date.
In the United States, more than 20,000 individuals are diagnosed with stomach (gastric) cancer each year. The American Cancer Society estimates that there will be 22,710 new cases of colorectal cancer diagnosed in the U.S. in 2004 (Cancer Facts and Figures 2004. Atlanta, Ga.: American Cancer Society, 2004. Available at www.cancer.org/docroot/STT/stt—0—2004.asp, accessed Aug. 21, 2005). Because stomach cancer may occur without symptoms, it may be in advanced stages by the time the diagnosis is made. Treatment is then directed at making the patient more comfortable and improving quality of life.
Lung cancer causes more deaths worldwide than any other form of cancer (Goodman, G. E., Thorax 57:994-999 (2002)). In the United States, lung cancer is the primary cause of cancer death among both men and women. In 2002, the death rate from lung cancer was an estimated 134,900 deaths, exceeding the combined total for breast, prostate and colon cancer. Id. Lung cancer is also the leading cause of cancer death in all European countries, and numbers of lung cancer-related deaths are rapidly increasing in developing countries as well.
The five-year survival rate among all lung cancer patients, regardless of the stage of disease at diagnosis, is only about 13%. This contrasts with a five-year survival rate of 46% among cases detected while the disease is still localized. However, only 16% of lung cancers are discovered before the disease has spread. Early detection is difficult as clinical symptoms are often not observed until the disease has reached an advanced stage. Despite research into therapies for this and other cancers, lung cancer remains difficult to diagnose and treat effectively.
Clearly, the identification of markers and genes that are responsible for susceptibility to particular forms of solid cancer (e.g., prostate cancer, breast cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer) is one of the major challenges facing oncology today. There is a need to identify means for the early detection of individuals that have a genetic susceptibility to cancer so that more aggressive screening and intervention regimens may be instituted for the early detection and treatment of cancer. Cancer genes may also reveal key molecular pathways that may be manipulated (e.g., using small or large molecule weight drugs) and may lead to more effective treatments regardless of the cancer stage when a particular cancer is first diagnosed.
MicroRNAs are a class of small, non-coding RNAs that control gene expression by hybridizing to and triggering either translational repression or, less frequently, degradation of a messenger RNA (mRNA) target. The discovery and study of miRNAs has revealed miRNA-mediated gene regulatory mechanisms that play important roles in organismal development and various cellular processes, such as cell differentiation, cell growth and cell death (Cheng, A. M., et al., Nucleic Acids Res. 33:1290-1297 (2005)). Recent studies suggest that aberrant expression of particular miRNAs may be involved in human diseases, such as neurological disorders (Ishizuka, A., et al., Genes Dev. 16:2497-2508 (2002)) and cancer. In particular, misexpression of miR-16-1 and/or miR-15a has been found in human chronic lymphocytic leukemias (Calin, G. A., et al., Proc. Natl. Acad. Sci. U.S.A. 99:15524-15529 (2002)).
Clearly, there is a great need in the art for improved methods for detecting and treating solid cancers (e.g., prostate cancer, breast cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer). The present invention provides novel methods and compositions for the diagnosis and treatment of solid cancers.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the identification of specific miRNAs that have altered expression levels in particular solid cancers.
Accordingly, the invention encompasses methods of diagnosing whether a subject has, or is at risk for developing, a solid cancer. According to the methods of the invention, the level of at least one miR gene product in a test sample from the subject is compared to the level of a corresponding miR gene product in a control sample. An alteration (e.g., an increase, a decrease) in the level of the miR gene product in the test sample, relative to the level of a corresponding miR gene product in a control sample, is indicative of the subject either having, or being at risk for developing, a solid cancer. The solid cancer can be any cancer that arises from organs and solid tissues. In certain embodiments, the solid cancer is stomach cancer, breast cancer, pancreatic cancer, colon cancer, lung cancer or prostate cancer. In particular embodiments, the solid cancer is not breast cancer, lung cancer, prostate cancer, pancreatic cancer or gastrointestinal cancer.
In one embodiment, the at least one miR gene product measured in the test sample is selected from the group consisting of miR-21, miR-191, miR-17-5p and combinations thereof. In another embodiment, the at least one miR gene product measured in the test sample is selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
In one embodiment, the solid cancer is breast cancer or lung cancer and the at least one miR gene product measured in the test sample is selected from the group consisting of miR-210, miR-213 and a combination thereof.
In another embodiment, the solid cancer is colon cancer, stomach cancer, prostate cancer or pancreas cancer and the at least one miR gene product measured in the test sample is miR-218-2.
In a certain embodiment, the solid cancer is breast cancer and the at least one miR gene product measured in the test sample is selected from the group consisting of miR-125b-1, miR-125b-2, miR-145, miR-21 and combinations thereof. In a related embodiment, the solid cancer is breast cancer and the at least one miR gene product in the test sample is selected from the group consisting of miR-21, miR-29b-2, miR-146, miR-125b-2, miR-125b-1, miR-10b, miR-145, miR-181a, miR-140, miR-213, miR-29a prec, miR-181b-1, miR-199b, miR-29b-1, miR-130a, miR-155, let-7a-2, miR-205, miR-29c, miR-224, miR-100, miR-31, miR-30c, miR-17-5p, miR-210, miR-122a, miR-16-2 and combinations thereof.
In another embodiment, the solid cancer is colon cancer and the at least one miR gene product in the test sample is selected from the group consisting of miR-24-1, miR-29b-2, miR-20a, miR-10a, miR-32, miR-203, miR-106a, miR-17-5p, miR-30c, miR-223, miR-126*, miR-128b, miR-21, miR-24-2, miR-99b prec, miR-155, miR-213, miR-150, miR-107, miR-191, miR-221, miR-9-3 and combinations thereof.
In yet another embodiment, the solid cancer is lung cancer and the miR gene product in the test sample is selected from the group consisting of miR-21, miR-205, miR-200b, miR-9-1, miR-210, miR-148, miR-141, miR-132, miR-215, miR-128b, let-7g, miR-16-2, miR-129-1/2 prec, miR-126*, miR-142-as, miR-30d, miR-30a-5p, miR-7-2, miR-199a-1, miR-127, miR-34a prec, miR-34a, miR-136, miR-202, miR-196-2, miR-199a-2, let-7a-2, miR-124a-1, miR-149, miR-17-5p, miR-196-1 prec, miR-10a, miR-99b prec, miR-196-1, miR-199b, miR-191, miR-195, miR-155 and combinations thereof.
In an additional embodiment, the solid cancer is pancreatic cancer and the at least one miR gene product measured in the test sample is selected from the group consisting of miR-103-1, miR-103-2, miR-155, miR-204 and combinations thereof. In a related embodiment, the solid cancer is pancreatic cancer and the miR gene product in the test sample is selected from the group consisting of miR-103-2, miR-103-1, miR-24-2, miR-107, miR-100, miR-125b-2, miR-125b-1, miR-24-1, miR-191, miR-23a, miR-26a-1, miR-125a, miR-130a, miR-26b, miR-145, miR-221, miR-126*, miR-16-2, miR-146, miR-214, miR-99b, miR-128b, miR-155, miR-29b-2, miR-29a, miR-25, miR-16-1, miR-99a, miR-224, miR-30d, miR-92-2, miR-199a-1, miR-223, miR-29c, miR-30b, miR-129-1/2, miR-197, miR-17-5p, miR-30c, miR-7-1, miR-93-1, miR-140, miR-30a-5p, miR-132, miR-181b-1, miR-152 prec, miR-23b, miR-20a, miR-222, miR-27a, miR-92-1, miR-21, miR-129-1/2 prec, miR-150, miR-32, miR-106a, miR-29b-1 and combinations thereof.
In another embodiment, the solid cancer is prostate cancer and the miR gene product in the test sample is selected from the group consisting of let-7d, miR-128a prec, miR-195, miR-203, let-7a-2 prec, miR-34a, miR-20a, miR-218-2, miR-29a, miR-25, miR-95, miR-197, miR-135-2, miR-187, miR-196-1, miR-148, miR-191, miR-21, let-71, miR-198, miR-199a-2, miR-30c, miR-17-5p, miR-92-2, miR-146, miR-181b-1 prec, miR-32, miR-206, miR-184 prec, miR-29a prec, miR-29b-2, miR-149, miR-181b-1, miR-196-1 prec, miR-93-1, miR-223, miR-16-1, miR-101-1, miR-124a-1, miR-26a-1, miR-214, miR-27a, miR-24-1, miR-106a, miR-199a-1 and combinations thereof.
In yet another embodiment, the solid cancer is stomach cancer and the miR gene product in the test sample is selected from the group consisting of miR-223, miR-21, miR-218-2, miR-103-2, miR-92-2, miR-25, miR-136, miR-191, miR-221, miR-125b-2, miR-103-1, miR-214, miR-222, miR-212 prec, miR-125b-1, miR-100, miR-107, miR-92-1, miR-96, miR-192, miR-23a, miR-215, miR-7-2, miR-138-2, miR-24-1, miR-99b, miR-33b, miR-24-2 and combinations thereof.
The level of the at least one miR gene product can be measured using a variety of techniques that are well known to those of skill in the art (e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis, solution hybridization detection). In a particular embodiment, the level of at least one miR gene product is measured by reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to one or more miRNA-specific probe oligonucleotides (e.g., hybridzing to a microarray that comprises several miRNA-specific probe oligonucleotides) to provide a hybridization profile for the test sample, and comparing the test sample hybridization profile to a hybridization profile from a control sample. An alteration in the signal of at least one miRNA in the test sample relative to the control sample is indicative of the subject either having, or being at risk for developing, a solid cancer. In a particular embodiment, target oligonucleotides are hybridized to a microarray comprising miRNA-specific probe oligonucleotides for one or more miRNAs selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
The invention also encompasses methods of inhibiting tumorigenesis in a subject who has, or is suspected of having, a solid cancer (e.g., prostate cancer, stomach cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer), wherein at least one miR gene product is deregulated (e.g., down-regulated, up-regulated) in the cancer cells of the subject. When the at least one isolated miR gene product is down-regulated in the cancer cells, the method comprises administering an effective amount of an isolated miR gene product, an isolated variant or a biologically-active fragment of the miR gene product or variant, such that proliferation of cancer cells in the subject is inhibited. In a further embodiment, the at least one isolated miR gene product is selected from the group consisting of miR-145, miR-155, miR-218-2 and combinations thereof. In a particular embodiment, the miR gene product is not miR-15a or miR-16-1. When the at least one isolated miR gene product is up-regulated in the cancer cells, the method comprises administering to the subject an effective amount of at least one compound for inhibiting expression of the at least one miR gene product (referred to herein as a “miR expression-inhibition compound”), such that proliferation of cancer cells in the subject is inhibited. In a particular embodiment, the at least one miR expression-inhibition compound is specific for a miR gene product selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
In a related embodiment, the methods of inhibiting tumorigenesis in a subject additionally comprise the step of determining the amount of at least one miR gene product in cancer cells from the subject, and comparing that level of the miR gene product in the cells to the level of a corresponding miR gene product in control cells. If expression of the miR gene product is deregulated (e.g., down-regulated, up-regulated) in cancer cells, the methods further comprise altering the amount of the at least one miR gene product expressed in the cancer cells. In one embodiment, the amount of the miR gene product expressed in the cancer cells is less than the amount of the miR gene product expressed in a control cell (e.g., control cells), and an effective amount of the down-regulated miR gene product, isolated variant or biologically-active fragment of the miR gene product or variant, is administered to the subject. Suitable miR gene products for this embodiment include miR-145, miR-155, miR-218-2 and combinations thereof, among others. In a particular embodiment, the miR gene product is not miR-15a or miR-16-1. In another embodiment, the amount of the miR gene product expressed in the cancer cells is greater than the amount of the miR gene product expressed in the control cell (e.g., control cells), and an effective amount of at least one compound for inhibiting expression of the at least one up-regulated miR gene product is administered to the subject. Suitable compounds for inhibiting expression of the at least one miR gene product include, but are not limited to, compounds that inhibit the expression of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
The invention further provides pharmaceutical compositions for treating solid cancers (e.g., prostate cancer, stomach cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer). In one embodiment, the pharmaceutical compositions comprise at least one isolated miR gene product and a pharmaceutically-acceptable carrier. In a particular embodiment, the at least one miR gene product corresponds to a miR gene product that has a decreased level of expression in cancer cells relative to control cells. In certain embodiments the isolated miR gene product is selected from the group consisting of miR-145, miR-155, miR-218-2 and combinations thereof.
In another embodiment, the pharmaceutical compositions of the invention comprise at least one miR expression-inhibition compound and a pharmaceutically-acceptable carrier. In a particular embodiment, the at least one miR expression-inhibition compound is specific for a miR gene product whose expression is greater in cancer cells than in control cells. In certain embodiments, the miR expression-inhibition compound is specific for one or more miR gene products selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
The invention also encompasses methods of identifying an inhibitor of tumorigenesis, comprising providing a test agent to a cell and measuring the level of at least one miR gene product in the cell. In one embodiment, the method comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with decreased expression levels in solid cancers (e.g., prostate cancer, stomach cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer). An increase in the level of the miR gene product in the cell, relative to a suitable control cell, is indicative of the test agent being an inhibitor of tumorigenesis. In a particular embodiment, the at least one miR gene product associated with decreased expression levels in solid cancer cells is selected from the group consisting of miR-145, miR-155, miR-218-2 and combinations thereof.
In other embodiments, the method comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with increased expression levels in solid cancers. A decrease in the level of the miR gene product in the cell, relative to a suitable control cell, is indicative of the test agent being an inhibitor of tumorigenesis. In a particular embodiment, the at least one miR gene product associated with increased expression levels in solid cancer cells is selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 depicts a clustering analysis of 540 samples, representing 6 solid cancers (top) and the respective normal tissues. miRNAs included in the tree (n=137) represent those whose expression level (background-subtracted intensity) was higher than the threshold value (256) in at least 50% of the samples analyzed. Arrays were median-centered and normalized using Gene Cluster 2.0. Average linkage clustering was performed by using uncentered correlation metric. The colors indicate the difference in expression level from the median for the microRNAs in each sample.
FIG. 2 depicts unsupervised analysis of microRNA expression data. MicroRNA profiling of 540 samples (indicated at top of panel) covering breast, colon, lung, pancreas, prostate and stomach (normal tissues and tumors) were filtered, centered and normalized for each feature. The data were subject to hierarchical clustering on both the samples (horizontally-oriented) and the features (vertically-oriented with average linkage and Pearson correlation as a similarity measure. Sample names are indicated at the top of the figure and miRNA names on the left. The probe ID is indicated in parentheses, as the same microRNA can be measured by different oligonucleotides. The colors indicate the difference in expression level from the median for the microRNAs in each sample.
FIG. 3 depicts the expression of differentially-regulated miRNAs across solid cancers (top). Sixty-one microRNAs, which are present in at least 90% of the tissues solid cancers, are represented (right of panel). The tree displays the average absolute expression values for each of the listed microRNAs after log2 transformation. The mean was computed over all samples from the same tissue or tumor histotype. Genes were mean-centered and normalized using Gene Cluster 2.0. Average linkage'clustering was performed using Euclidean distance.
FIG. 4 depicts fold changes in the expression of miRNAs present in at least 75% of the solid tumors with at least 1 tumor absolute value higher than 2 in different cancer samples (top), relative to normal samples. The tree displays the log2 transformation of average fold changes (cancer vs. normal). The mean was computed over all samples from the same tissue or tumor histotype. Arrays were mean-centered and normalized using Gene Cluster 2.0. Average linkage clustering was performed using uncentered correlation metric.
FIG. 5 depicts fold changes in the expression of miRNAs present in the signatures of at least 50% of the solid tumors in cancer vs. normal samples. The tree displays the log2 transformation of the average fold changes (cancer over normal). The mean was computed over all samples from the same tissue or tumor histotype. Arrays were mean centered and normalized using Gene Cluster 2.0. Average linkage clustering was performed using uncentered correlation metric.
FIG. 6A depicts bar graphs indicating that the 3′UTR of different genes encoding cancer protein enables cancer regulation by microRNA. The relative repression of firefly luciferase expression (Fold Change) standardized to a renilla luciferase control. PLAG1, pleiomorphic adenoma gene 1; TGFBR2, transforming growth factor beta receptor II; Rb, retinoblastoma gene. pGL-3 (Promega) was used as the empty vector. miR-20a, miR-26a-1 and miR-106 oligoRNAs (sense and scrambled) were used for transfections. A second experiment using mutated versions of each target mRNA, which lack the 5′ miRNA-end complementarity site (MUT), as controls is shown in the bottom panel. All the experiments were performed twice in triplicate (n=6).
FIG. 6B depicts Western blots indicating that, in certain cancers (e.g., lung, breast, colon, gastric), the levels of RB1 (Rb) protein displays an inverse correlation with the level of miR-106a expression. β-Actin was used as a control for normalization. N1, normal sample; T1 and T2, tumor sample.
FIG. 7 depicts Northern blots showing down-regulation of miR-145 (top) and up-regulation of miR-21 (bottom) expression in breast cancer samples (P series and numbered series) relative to normal samples. Normalization was performed with a U6-specific probe.
FIG. 8 depicts Northern blots showing up-regulation of miR-103 and down-regulation miR-155 (top) expression in different endocrine pancreatic cancer samples (WDET, well differentiated pancreatic endocrine tumors, WDEC, well differentiated pancreatic endocrine carcinomas and ACC, pancreatic acinar cell carcinomas) relative to normal samples (K series), as well as up-regulation of miR-204 (bottom) expression in insulinomas (F series) relative to normal samples (K series) and non secreting/non functioning (NF-series) samples. Normalization was performed with a probe specific to 5S RNA.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, in part, on the identification of particular microRNAs whose expression is altered in cancer cells associated with different solid cancers, such as colon, stomach, pancreatic, lung, breast and prostate cancer, relative to normal control cells.
As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,” or “miRNA” refers to the unprocessed (e.g., precursor) or processed (e.g., mature) RNA transcript from a miR gene. As the miR gene products are not translated into protein, the term “miR gene products” does not include proteins. The unprocessed miR gene transcript is also called a “miR precursor” or “miR prec” and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miR precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III (e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the “processed” miR gene transcript or “mature” miRNA.
The active 19-25 nucleotide RNA molecule can be obtained from the miR precursor through natural processing routes (e.g., using intact cells or cell lysates) or by synthetic processing routes (e.g., using isolated processing enzymes, such as isolated Dicer, Argonaut, or RNAse III). It is understood that the active 19-25 nucleotide RNA molecule can also be produced directly by biological or chemical synthesis, without having been processed from the miR precursor. When a microRNA is referred to herein by name, the name corresponds to both the precursor and mature forms, unless otherwise indicated.
Tables 1a and 1b depict the nucleotide sequences of particular precursor and mature human microRNAs.







TABLE 1a







Human microRNA Precursor Sequences











SEQ


Precursor

ID


Name
Sequence (5′ To 3′)*
NO.












let-7a-1
CACUGUGGGAUGAGGUAGUAGGUUGUAUAGUUU
1



UAGGGUCACACCCACCACUGGGAGAUAACUAUA



CAAUCUACUGUCUUUCCUAACGUG


 


let-7a-2
AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUU
2



ACAUCAAGGGAGAUAACUGUACAGCCUCCUAGC



UUUCCU


 


let-7a-3
GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUC
3



UGCCCUGCUAUGGGAUAACUAUACAAUCUACUG



UCUUUCCU


 


let-7a-4
GUGACUGCAUGCUCCCAGGUUGAGGUAGUAGGU
4



UGUAUAGUUUAGAAUUACACAAGGGAGAUAACU



GUACAGCCUCCUAGCUUUCCUUGGGUCUUGCAC



UAAACAAC


 


let-7b
GGCGGGGUGAGGUAGUAGGUUGUGUGGUUUCAG
5



GGCAGUGAUGUUGCCCCUCGGAAGAUAACUAUA



CAACCUACUGCCUUCCCUG


 


let-7c
GCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUU
6



AGAGUUACACCCUGGGAGUUAACUGUACAACCU



UCUAGCUUUCCUUGGAGC


 


let-7d
CCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAG
7



GGCAGGGAUUUUGCCCACAAGGAGGUAACUAUA



CGACCUGCUGCCUUUCUUAGG


 


let-7d-v1
CUAGGAAGAGGUAGUAGUUUGCAUAGUUUUAGG
8



GCAAAGAUUUUGCCCACAAGUAGUUAGCUAUAC



GACCUGCAGCCUUUUGUAG


 


let-7d-v2
CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGG
9



GUUGUGACAUUGCCCGCUGUGGAGAUAACUGCG



CAAGCUACUGCCUUGCUAG


 


let-7e
CCCGGGCUGAGGUAGGAGGUUGUAUAGUUGAGG
10



AGGACACCCAAGGAGAUCACUAUACGGCCUCCU



AGCUUUCCCCAGG


 


let-7f-1
UCAGAGUGAGGUAGUAGAUUGUAUAGUUGUGGG
11



GUAGUGAUUUUACCCUGUUCAGGAGAUAACUAU



ACAAUCUAUUGCCUUCCCUGA


 


let-7f-2-1
CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUGUG
12



GGGUAGUGAUUUUACCCUGUUCAGGAGAUAACU



AUACAAUCUAUUGCCUUCCCUGA


 


let-7f-2-2
CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUUUA
13



GGGUCAUACCCCAUCUUGGAGAUAACUAUACAG



UCUACUGUCUUUCCCACGG


 


let-7g
UUGCCUGAUUCCAGGCUGAGGUAGUAGUUUGUA
14



CAGUUUGAGGGUCUAUGAUACCACCCGGUACAG



GAGAUAACUGUACAGGCCACUGCCUUGCCAGGA



ACAGCGCGC


 


let-7i
CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGG
15



GUUGUGACAUUGCCCGCUGUGGAGAUAACUGCG



CAAGCUACUGCCUUGCUAG


 


miR-1b-1-1
ACCUACUCAGAGUACAUACUUCUUUAUGUACCC
16



AUAUGAACAUACAAUGCUAUGGAAUGUAAAGAA



GUAUGUAUUUUUGGUAGGC


 


miR-1b-1-2
CAGCUAACAACUUAGUAAUACCUACUCAGAGUA
17



CAUACUUCUUUAUGUACCCAUAUGAACAUACAA



UGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGG



UAGGCAAUA


 


miR-1b-2
GCCUGCUUGGGAAACAUACUUCUUUAUAUGCCC
18



AUAUGGACCUGCUAAGCUAUGGAAUGUAAAGAA



GUAUGUAUCUCAGGCCGGG


 


miR-1b
UGGGAAACAUACUUCUUUAUAUGCCCAUAUGGA
19



CCUGCUAAGCUAUGGAAUGUAAAGAAGUAUGUA



UCUCA


 


miR-1d
ACCUACUCAGAGUACAUACUUCUUUAUGUACCC
20



AUAUGAACAUACAAUGCUAUGGAAUGUAAAGAA



GUAUGUAUUUUUGGUAGGC


 


miR-7-1a
UGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAG
21



UGAUUUUGUUGUUUUUAGAUAACUAAAUCGACA



ACAAAUCACAGUCUGCCAUAUGGCACAGGCCAU



GCCUCUACA


 


miR-7-1b
UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUA
22



GUGAUUUUGUUGUUUUUAGAUAACUAAAUCGAC



AACAAAUCACAGUCUGCCAUAUGGCACAGGCCA



UGCCUCUACAG


 


miR-7-2
CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUG
23



GAAGACUAGUGAUUUUGUUGUUGUCUUACUGCG



CUCAACAACAAAUCCCAGUCUACCUAAUGGUGC



CAGCCAUCGCA


 


miR-7-3
AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGG
24



AAGACUAGUGAUUUUGUUGUUCUGAUGUACUAC



GACAACAAGUCACAGCCGGCCUCAUAGCGCAGA



CUCCCUUCGAC


 


miR-9-1
CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUG
25



UAUGAGUGGUGUGGAGUCUUCAUAAAGCUAGAU



AACCGAAAGUAAAAAUAACCCCA


 


miR-9-2
GGAAGCGAGUUGUUAUCUUUGGUUAUCUAGCUG
26



UAUGAGUGUAUUGGUCUUCAUAAAGCUAGAUAA



CCGAAAGUAAAAACUCCUUCA


 


miR-9-3
GGAGGCCCGUUUCUCUCUUUGGUUAUCUAGCUG
27



UAUGAGUGCCACAGAGCCGUCAUAAAGCUAGAU



AACCGAAAGUAGAAAUGAUUCUCA


 


miR-10a
GAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAU
28



CCGAAUUUGUGUAAGGAAUUUUGUGGUCACAAA



UUCGUAUCUAGGGGAAUAUGUAGUUGACAUAAA



CACUCCGCUCU


 


miR-10b
CCAGAGGUUGUAACGUUGUCUAUAUAUACCCUG
29



UAGAACCGAAUUUGUGUGGUAUCCGUAUAGUCA



CAGAUUCGAUUCUAGGGGAAUAUAUGGUCGAUG



CAAAAACUUCA


 


miR-15a-2
GCGCGAAUGUGUGUUUAAAAAAAAUAAAACCUU
30



GGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGA



UUUUGAAAAGGUGCAGGCCAUAUUGUGCUGCCU



CAAAAAUAC


 


miR-15a
CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUG
31



UGGAUUUUGAAAAGGUGCAGGCCAUAUUGUGCU



GCCUCAAAAAUACAAGG


 


miR-15b-1
CUGUAGCAGCACAUCAUGGUUUACAUGCUACAG
32



UCAAGAUGCGAAUCAUUAUUUGCUGCUCUAG


 


miR-15b-2
UUGAGGCCUUAAAGUACUGUAGCAGCACAUCAU
33



GGUUUACAUGCUACAGUCAAGAUGCGAAUCAUU



AUUUGCUGCUCUAGAAAUUUAAGGAAAUUCAU


 


miR-16-1
GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGG
34



CGUUAAGAUUCUAAAAUUAUCUCCAGUAUUAAC



UGUGCUGCUGAAGUAAGGUUGAC


 


miR-16-2
GUUCCACUCUAGCAGCACGUAAAUAUUGGCGUA
35



GUGAAAUAUAUAUUAAACACCAAUAUUACUGUG



CUGCUUUAGUGUGAC


 


miR-16-13
GCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUU
36



AAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUG



CUGCUGAAGUAAGGU


 


miR-17
GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGG
37



UAGUGAUAUGUGCAUCUACUGCAGUGAAGGCAC



UUGUAGCAUUAUGGUGAC


 


miR-18
UGUUCUAAGGUGCAUCUAGUGCAGAUAGUGAAG
38



UAGAUUAGCAUCUACUGCCCUAAGUGCUCCUUC



UGGCA


 


miR-18-13
UUUUUGUUCUAAGGUGCAUCUAGUGCAGAUAGU
39



GAAGUAGAUUAGCAUCUACUGCCCUAAGUGCUC



CUUCUGGCAUAAGAA


 


miR-19a
GCAGUCCUCUGUUAGUUUUGCAUAGUUGCACUA
40



CAAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAA



ACUGAUGGUGGCCUGC


 


miR-19a-13
CAGUCCUCUGUUAGUUUUGCAUAGUUGCACUAC
41



AAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAAA



CUGAUGGUGGCCUG


 


miR-19b-1
CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAU
42



CCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAU



GCAAAACUGACUGUGGUAGUG


 


miR-19b-2
ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUG
43



CAUUUCAGCGUAUAUAUGUAUAUGUGGCUGUGC



AAAUCCAUGCAAAACUGAUUGUGAUAAUGU


 


miR-19b-13
UUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGC
44



UGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAA



ACUGACUGUGGUAG


 


miR-19b-X
UUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCG
45



UAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGC



AAAACUGAUUGUGAU


 


miR-20
GUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGU
46


(miR-20a)
UUAGUUAUCUACUGCAUUAUGAGCACUUAAAGU



ACUGC


 


miR-21
UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGU
47



UGAAUCUCAUGGCAACACCAGUCGAUGGGCUGU



CUGACA


 


miR-21-17
ACCUUGUCGGGUAGCUUAUCAGACUGAUGUUGA
48



CUGUUGAAUCUCAUGGCAACACCAGUCGAUGGG



CUGUCUGACAUUUUG


 


miR-22
GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCU
49



UUAUGUCCUGACCCAGCUAAAGCUGCCAGUUGA



AGAACUGUUGCCCUCUGCC


 


miR-23a
GGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCU
50



UCCUGUCACAAAUCACAUUGCCAGGGAUUUCCA



ACCGACC


 


miR-23b
CUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAU
51



GCUGAUUUGUGACUUAAGAUUAAAAUCACAUUG



CCAGGGAUUACCACGCAACCACGACCUUGGC


 


miR-23-19
CCACGGCCGGCUGGGGUUCCUGGGGAUGGGAUU
52



UGCUUCCUGUCACAAAUCACAUUGCCAGGGAUU



UCCAACCGACCCUGA


 


miR-24-1
CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCA
53



UUUUACACACUGGCUCAGUUCAGCAGGAACAGG



AG


 


miR-24-2
CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAG
54



UUGGUUUGUGUACACUGGCUCAGUUCAGCAGGA



ACAGGG


 


miR-24-19
CCCUGGGCUCUGCCUCCCGUGCCUACUGAGCUGA
55



AACACAGUUGGUUUGUGUACACUGGCUCAGUUC



AGCAGGAACAGGGG


 


miR-24-9
CCCUCCGGUGCCUACUGAGCUGAUAUCAGUUCU
56



CAUUUUACACACUGGCUCAGUUCAGCAGGAACA



GCAUC


 


miR-25
GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUU
57



GCUGGACGCUGCCCUGGGCAUUGCACUUGUCUC



GGUCUGACAGUGCCGGCC


 


miR-26a
AGGCCGUGGCCUCGUUCAAGUAAUCCAGGAUAG
58



GCUGUGCAGGUCCCAAUGGCCUAUCUUGGUUAC



UUGCACGGGGACGCGGGCCU


 


miR-26a-1
GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGU
59



GCAGGUCCCAAUGGGCCUAUUCUUGGUUACUUG



CACGGGGACGC


 


miR-26a-2
GGCUGUGGCUGGAUUCAAGUAAUCCAGGAUAGG
60



CUGUUUCCAUCUGUGAGGCCUAUUCUUGAUUAC



UUGUUUCUGGAGGCAGCU


 


miR-26b
CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUU
61



GUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGC



UCGGGGACCGG


 


miR-27a
CUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGG
62



GUCCACACCAAGUCGUGUUCACAGUGGCUAAGU



UCCGCCCCCCAG


 


miR-27b-1
AGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGA
63



UUGGUUUCCGCUUUGUUCACAGUGGCUAAGUUC



UGCACCU


 


miR-27b-2
ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUU
64



GGUGAACAGUGAUUGGUUUCCGCUUUGUUCACA



GUGGCUAAGUUCUGCACCUGAAGAGAAGGUG


 


miR-27-19
CCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAG
65



GGUCCACACCAAGUCGUGUUCACAGUGGCUAAG



UUCCGCCCCCCAGG


 


miR-28
GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUG
66



AGUUACCUUUCUGACUUUCCCACUAGAUUGUGA



GCUCCUGGAGGGCAGGCACU


 


miR-29a-2
CCUUCUGUGACCCCUUAGAGGAUGACUGAUUUC
67



UUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGC



ACCAUCUGAAAUCGGUUAUAAUGAUUGGGGAAG



AGCACCAUG


 


miR-29a
AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUA
68



UAAUUUUCUAGCACCAUCUGAAAUCGGUUAU


 


miR-29b-1
CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGA
69



UUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAU



CAGUGUUCUUGGGGG


 


miR-29b-2
CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGA
70



UUUUUCCAUCUUUGUAUCUAGCACCAUUUGAAA



UCAGUGUUUUAGGAG


 


miR-29c
ACCACUGGCCCAUCUCUUACACAGGCUGACCGAU
71



UUCUCCUGGUGUUCAGAGUCUGUUUUUGUCUAG



CACCAUUUGAAAUCGGUUAUGAUGUAGGGGGAA



AAGCAGCAGC


 


miR-30a
GCGACUGUAAACAUCCUCGACUGGAAGCUGUGA
72



AGCCACAGAUGGGCUUUCAGUCGGAUGUUUGCA



GCUGC


 


miR-30b-1
AUGUAAACAUCCUACACUCAGCUGUAAUACAUG
73



GAUUGGCUGGGAGGUGGAUGUUUACGU


 


miR-30b-2
ACCAAGUUUCAGUUCAUGUAAACAUCCUACACU
74



CAGCUGUAAUACAUGGAUUGGCUGGGAGGUGGA



UGUUUACUUCAGCUGACUUGGA


 


miR-30c
AGAUACUGUAAACAUCCUACACUCUCAGCUGUG
75



GAAAGUAAGAAAGCUGGGAGAAGGCUGUUUACU



CUUUCU


 


miR-30d
GUUGUUGUAAACAUCCCCGACUGGAAGCUGUAA
76



GACACAGCUAAGCUUUCAGUCAGAUGUUUGCUG



CUAC


 


miR-30e
CUGUAAACAUCCUUGACUGGAAGCUGUAAGGUG
77



UUCAGAGGAGCUUUCAGUCGGAUGUUUACAG


 


miR-31
GGAGAGGAGGCAAGAUGCUGGCAUAGCUGUUGA
78



ACUGGGAACCUGCUAUGCCAACAUAUUGCCAUC



UUUCC


 


miR-32
GGAGAUAUUGCACAUUACUAAGUUGCAUGUUGU
79



CACGGCCUCAAUGCAAUUUAGUGUGUGUGAUAU



UUUC


 


miR-33b
GGGGGCCGAGAGAGGCGGGCGGCCCCGCGGUGC
80



AUUGCUGUUGCAUUGCACGUGUGUGAGGCGGGU



GCAGUGCCUCGGCAGUGCAGCCCGGAGCCGGCCC



CUGGCACCAC


 


miR-33b-2
ACCAAGUUUCAGUUCAUGUAAACAUCCUACACU
81



CAGCUGUAAUACAUGGAUUGGCUGGGAGGUGGA



UGUUUACUUCAGCUGACUUGGA


 


miR-33
CUGUGGUGCAUUGUAGUUGCAUUGCAUGUUCUG
82



GUGGUACCCAUGCAAUGUUUCCACAGUGCAUCA



CAG


 


miR-34-a
GGCCAGCUGUGAGUGUUUCUUUGGCAGUGUCUU
83



AGCUGGUUGUUGUGAGCAAUAGUAAGGAAGCAA



UCAGCAAGUAUACUGCCCUAGAAGUGCUGCACG



UUGUGGGGCCC


 


miR-34-b
GUGCUCGGUUUGUAGGCAGUGUCAUUAGCUGAU
84



UGUACUGUGGUGGUUACAAUCACUAACUCCACU



GCCAUCAAAACAAGGCAC


 


miR-34-c
AGUCUAGUUACUAGGCAGUGUAGUUAGCUGAUU
85



GCUAAUAGUACCAAUCACUAACCACACGGCCAG



GUAAAAAGAUU


 


miR-91-13
UCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGU
86



AGUGAUAUGUGCAUCUACUGCAGUGAAGGCACU



UGUAGCAUUAUGGUGA


 


miR-92-1
CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCU
87



GUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCC



UGUUGAGUUUGG


 


miR-92-2
UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUG
88



UGUUCUAUAUAAAGUAUUGCACUUGUCCCGGCC



UGUGGAAGA


 


miR-93-1
CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAG
89


(miR-93-2)
UGUGAUUACCCAACCUACUGCUGAGCUAGCACU



UCCCGAGCCCCCGG


 


miR-95-4
AACACAGUGGGCACUCAAUAAAUGUCUGUUGAA
90



UUGAAAUGCGUUACAUUCAACGGGUAUUUAUUG



AGCACCCACUCUGUG


 


miR-96-7
UGGCCGAUUUUGGCACUAGCACAUUUUUGCUUG
91



UGUCUCUCCGCUCUGAGCAAUCAUGUGCAGUGC



CAAUAUGGGAAA


 


miR-97-6
GUGAGCGACUGUAAACAUCCUCGACUGGAAGCU
92


(miR-30*)
GUGAAGCCACAGAUGGGCUUUCAGUCGGAUGUU



UGCAGCUGCCUACU


 


miR-98
GUGAGGUAGUAAGUUGUAUUGUUGUGGGGUAGG
93



GAUAUUAGGCCCCAAUUAGAAGAUAACUAUACA



ACUUACUACUUUCC


 


miR-99b
GGCACCCACCCGUAGAACCGACCUUGCGGGGCCU
94



UCGCCGCACACAAGCUCGUGUCUGUGGGUCCGU



GUC


 


miR-99a
CCCAUUGGCAUAAACCCGUAGAUCCGAUCUUGU
95



GGUGAAGUGGACCGCACAAGCUCGCUUCUAUGG



GUCUGUGUCAGUGUG


 


miR-100-1/2
AAGAGAGAAGAUAUUGAGGCCUGUUGCCACAAA
96



CCCGUAGAUCCGAACUUGUGGUAUUAGUCCGCA



CAAGCUUGUAUCUAUAGGUAUGUGUCUGUUAGG



CAAUCUCAC


 


miR-100-11
CCUGUUGCCACAAACCCGUAGAUCCGAACUUGU
97



GGUAUUAGUCCGCACAAGCUUGUAUCUAUAGGU



AUGUGUCUGUUAGG


 


miR-101-1/2
AGGCUGCCCUGGCUCAGUUAUCACAGUGCUGAU
98



GCUGUCUAUUCUAAAGGUACAGUACUGUGAUAA



CUGAAGGAUGGCAGCCAUCUUACCUUCCAUCAG



AGGAGCCUCAC


 


miR-101
UCAGUUAUCACAGUGCUGAUGCUGUCCAUUCUA
99



AAGGUACAGUACUGUGAUAACUGA


 


miR-101-1
UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUG
100



UCUAUUCUAAAGGUACAGUACUGUGAUAACUGA



AGGAUGGCA


 


miR-101-2
ACUGUCCUUUUUCGGUUAUCAUGGUACCGAUGC
101



UGUAUAUCUGAAAGGUACAGUACUGUGAUAACU



GAAGAAUGGUGGU


 


miR-101-9
UGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUG
102



UAUAUCUGAAAGGUACAGUACUGUGAUAACUGA



AGAAUGGUG


 


miR-102-1
CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGA
103



UUUUUCCAUCUUUGUAUCUAGCACCAUUUGAAA



UCAGUGUUUUAGGAG


 


miR-102-7.1
CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGA
104


(miR-102-
UUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAU


7.2)
CAGUGUUCUUGGGGG


 


miR-103-2
UUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUG
105



UAGCAUUCAGGUCAAGCAACAUUGUACAGGGCU



AUGAAAGAACCA


 


miR-103-1
UACUGCCCUCGGCUUCUUUACAGUGCUGCCUUG
106



UUGCAUAUGGAUCAAGCAGCAUUGUACAGGGCU



AUGAAGGCAUUG


 


miR-104-17
AAAUGUCAGACAGCCCAUCGACUGGUGUUGCCA
107



UGAGAUUCAACAGUCAACAUCAGUCUGAUAAGC



UACCCGACAAGG


 


miR-105-1
UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUG
108



GUGGCUGCUCAUGCACCACGGAUGUUUGAGCAU



GUGCUACGGUGUCUA


 


miR-105-2
UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUG
109



GUGGCUGCUUAUGCACCACGGAUGUUUGAGCAU



GUGCUAUGGUGUCUA


 


miR-106-a
CCUUGGCCAUGUAAAAGUGCUUACAGUGCAGGU
110



AGCUUUUUGAGAUCUACUGCAAUGUAAGCACUU



CUUACAUUACCAUGG


 


miR-106-b
CCUGCCGGGGCUAAAGUGCUGACAGUGCAGAUA
111



GUGGUCCUCUCCGUGCUACCGCACUGUGGGUAC



UUGCUGCUCCAGCAGG


 


miR-107
CUCUCUGCUUUCAGCUUCUUUACAGUGUUGCCU
112



UGUGGCAUGGAGUUCAAGCAGCAUUGUACAGGG



CUAUCAAAGCACAGA


 


miR-108-1-
ACACUGCAAGAACAAUAAGGAUUUUUAGGGGCA
113


small
UUAUGACUGAGUCAGAAAACACAGCUGCCCCUG



AAAGUCCCUCAUUUUUCUUGCUGU


 


miR-108-2-
ACUGCAAGAGCAAUAAGGAUUUUUAGGGGCAUU
114


small
AUGAUAGUGGAAUGGAAACACAUCUGCCCCCAA



AAGUCCCUCAUUUU


 


miR-122a-1
CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGU
115



UUGUGUCUAAACUAUCAAACGCCAUUAUCACAC



UAAAUAGCUACUGCUAGGC


 


miR-122a-2
AGCUGUGGAGUGUGACAAUGGUGUUUGUGUCCA
116



AACUAUCAAACGCCAUUAUCACACUAAAUAGCU


 


miR-123
ACAUUAUUACUUUUGGUACGCGCUGUGACACUU
117



CAAACUCGUACCGUGAGUAAUAAUGCGC


 


miR-124a-1
AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUG
118



AUUUAAAUGUCCAUACAAUUAAGGCACGCGGUG



AAUGCCAAGAAUGGGGCUG


 


miR-124a-2
AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCAC
119



AGCGGACCUUGAUUUAAUGUCAUACAAUUAAGG



CACGCGGUGAAUGCCAAGAGCGGAGCCUACGGC



UGCACUUGAAG


 


miR-124a-3
UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUU
120



GAUUUAAUGUCUAUACAAUUAAGGCACGCGGUG



AAUGCCAAGAGAGGCGCCUCC


 


miR-124a
CUCUGCGUGUUCACAGCGGACCUUGAUUUAAUG
121



UCUAUACAAUUAAGGCACGCGGUGAAUGCCAAG



AG


 


miR-124b
CUCUCCGUGUUCACAGCGGACCUUGAUUUAAUG
122



UCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAG


 


miR-125a-1
UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACC
123



UGUGAGGACAUCCAGGGUCACAGGUGAGGUUCU



UGGGAGCCUGGCGUCUGGCC


 


miR-125a-2
GGUCCCUGAGACCCUUUAACCUGUGAGGACAUC
124



CAGGGUCACAGGUGAGGUUCUUGGGAGCCUGG


 


miR-125b-1
UGCGCUCCUCUCAGUCCCUGAGACCCUAACUUGU
125



GAUGUUUACCGUUUAAAUCCACGGGUUAGGCUC



UUGGGAGCUGCGAGUCGUGCU


 


miR-125b-2
ACCAGACUUUUCCUAGUCCCUGAGACCCUAACU
126



UGUGAGGUAUUUUAGUAACAUCACAAGUCAGGC



UCUUGGGACCUAGGCGGAGGGGA


 


miR-126-1
CGCUGGCGACGGGACAUUAUUACUUUUGGUACG
127



CGCUGUGACACUUCAAACUCGUACCGUGAGUAA



UAAUGCGCCGUCCACGGCA


 


miR-126-2
ACAUUAUUACUUUUGGUACGCGCUGUGACACUU
128



CAAACUCGUACCGUGAGUAAUAAUGCGC


 


miR-127-1
UGUGAUCACUGUCUCCAGCCUGCUGAAGCUCAG
129



AGGGCUCUGAUUCAGAAAGAUCAUCGGAUCCGU



CUGAGCUUGGCUGGUCGGAAGUCUCAUCAUC


 


miR-127-2
CCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCA
130



GAAAGAUCAUCGGAUCCGUCUGAGCUUGGCUGG



UCGG


 


miR-128a
UGAGCUGUUGGAUUCGGGGCCGUAGCACUGUCU
131



GAGAGGUUUACAUUUCUCACAGUGAACCGGUCU



CUUUUUCAGCUGCUUC


 


miR-128b
GCCCGGCAGCCACUGUGCAGUGGGAAGGGGGGC
132



CGAUACACUGUACGAGAGUGAGUAGCAGGUCUC



ACAGUGAACCGGUCUCUUUCCCUACUGUGUCAC



ACUCCUAAUGG


 


miR-128
GUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGG
133



UUUACAUUUCUCACAGUGAACCGGUCUCUUUUU



CAGC


 


miR-129-1
UGGAUCUUUUUGCGGUCUGGGCUUGCUGUUCCU
134



CUCAACAGUAGUCAGGAAGCCCUUACCCCAAAA



AGUAUCUA


 


miR-129-2
UGCCCUUCGCGAAUCUUUUUGCGGUCUGGGCUU
135



GCUGUACAUAACUCAAUAGCCGGAAGCCCUUAC



CCCAAAAAGCAUUUGCGGAGGGCG


 


miR-130a
UGCUGCUGGCCAGAGCUCUUUUCACAUUGUGCU
136



ACUGUCUGCACCUGUCACUAGCAGUGCAAUGUU



AAAAGGGCAUUGGCCGUGUAGUG


 


miR-131-1
GCCAGGAGGCGGGGUUGGUUGUUAUCUUUGGUU
137



AUCUAGCUGUAUGAGUGGUGUGGAGUCUUCAUA



AAGCUAGAUAACCGAAAGUAAAAAUAACCCCAU



ACACUGCGCAG


 


miR-131-3
CACGGCGCGGCAGCGGCACUGGCUAAGGGAGGC
138



CCGUUUCUCUCUUUGGUUAUCUAGCUGUAUGAG



UGCCACAGAGCCGUCAUAAAGCUAGAUAACCGA



AAGUAGAAAUG


 


miR-131
GUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUG
139



UAUUGGUCUUCAUAAAGCUAGAUAACCGAAAGU



AAAAAC


 


miR-132-1
CCGCCCCCGCGUCUCCAGGGCAACCGUGGCUUUC
140



GAUUGUUACUGUGGGAACUGGAGGUAACAGUCU



ACAGCCAUGGUCGCCCCGCAGCACGCCCACGCGC


 


miR-132-2
GGGCAACCGUGGCUUUCGAUUGUUACUGUGGGA
141



ACUGGAGGUAACAGUCUACAGCCAUGGUCGCCC


 


miR-133a-1
ACAAUGCUUUGCUAGAGCUGGUAAAAUGGAACC
142



AAAUCGCCUCUUCAAUGGAUUUGGUCCCCUUCA



ACCAGCUGUAGCUAUGCAUUGA


 


miR-133a-2
GGGAGCCAAAUGCUUUGCUAGAGCUGGUAAAAU
143



GGAACCAAAUCGACUGUCCAAUGGAUUUGGUCC



CCUUCAACCAGCUGUAGCUGUGCAUUGAUGGCG



CCG


 


miR-133
GCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUC
144



UUCAAUGGAUUUGGUCCCCUUCAACCAGCUGUA



GC


 


miR-133b
CCUCAGAAGAAAGAUGCCCCCUGCUCUGGCUGG
145



UCAAACGGAACCAAGUCCGUCUUCCUGAGAGGU



UUGGUCCCCUUCAACCAGCUACAGCAGGGCUGG



CAAUGCCCAGUCCUUGGAGA


 


miR-133b-
GCCCCCUGCUCUGGCUGGUCAAACGGAACCAAG
146


small
UCCGUCUUCCUGAGAGGUUUGGUCCCCUUCAAC



CAGCUACAGCAGGG


 


miR-134-1
CAGGGUGUGUGACUGGUUGACCAGAGGGGCAUG
147



CACUGUGUUCACCCUGUGGGCCACCUAGUCACCA



ACCCUC


 


miR-134-2
AGGGUGUGUGACUGGUUGACCAGAGGGGCAUGC
148



ACUGUGUUCACCCUGUGGGCCACCUAGUCACCA



ACCCU


 


miR-135a-1
AGGCCUCGCUGUUCUCUAUGGCUUUUUAUUCCU
149



AUGUGAUUCUACUGCUCACUCAUAUAGGGAUUG



GAGCCGUGGCGCACGGCGGGGACA


 


miR-135a-2
AGAUAAAUUCACUCUAGUGCUUUAUGGCUUUUU
150


(miR-135-2)
AUUCCUAUGUGAUAGUAAUAAAGUCUCAUGUAG



GGAUGGAAGCCAUGAAAUACAUUGUGAAAAAUCA


 


miR-135
CUAUGGCUUUUUAUUCCUAUGUGAUUCUACUGC
151



UCACUCAUAUAGGGAUUGGAGCCGUGG


 


miR-135b
CACUCUGCUGUGGCCUAUGGCUUUUCAUUCCUA
152



UGUGAUUGCUGUCCCAAACUCAUGUAGGGCUAA



AAGCCAUGGGCUACAGUGAGGGGCGAGCUCC


 


miR-136-1
UGAGCCCUCGGAGGACUCCAUUUGUUUUGAUGA
153



UGGAUUCUUAUGCUCCAUCAUCGUCUCAAAUGA



GUCUUCAGAGGGUUCU


 


miR-136-2
GAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUA
154



UGCUCCAUCAUCGUCUCAAAUGAGUCUUC


 


miR-137
CUUCGGUGACGGGUAUUCUUGGGUGGAUAAUAC
155



GGAUUACGUUGUUAUUGCUUAAGAAUACGCGUA



GUCGAGG


 


miR-138-1
CCCUGGCAUGGUGUGGUGGGGCAGCUGGUGUUG
156



UGAAUCAGGCCGUUGCCAAUCAGAGAACGGCUA



CUUCACAACACCAGGGCCACACCACACUACAGG


 


miR-138-2
CGUUGCUGCAGCUGGUGUUGUGAAUCAGGCCGA
157



CGAGCAGCGCAUCCUCUUACCCGGCUAUUUCACG



ACACCAGGGUUGCAUCA


 


miR-138
CAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGC
158



GCAUCCUCUUACCCGGCUAUUUCACGACACCAGG



GUUG


 


miR-139
GUGUAUUCUACAGUGCACGUGUCUCCAGUGUGG
159



CUCGGAGGCUGGAGACGCGGCCCUGUUGGAGUA



AC


 


miR-140
UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUA
160



CCCUAUGGUAGGUUACGUCAUGCUGUUCUACCA



CAGGGUAGAACCACGGACAGGAUACCGGGGCACC


 


miR-140as
UCCUGCCAGUGGUUUUACCCUAUGGUAGGUUAC
161



GUCAUGCUGUUCUACCACAGGGUAGAACCACGG



ACAGGA


 


miR-140s
CCUGCCAGUGGUUUUACCCUAUGGUAGGUUACG
162



UCAUGCUGUUCUACCACAGGGUAGAACCACGGA



CAGG


 


miR-141-1
CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUG
163



UUGGAUGGUCUAAUUGUGAAGCUCCUAACACUG



UCUGGUAAAGAUGGCUCCCGGGUGGGUUC


 


miR-141-2
GGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCU
164



AAUUGUGAAGCUCCUAACACUGUCUGGUAAAGA



UGGCCC


 


miR-142
ACCCAUAAAGUAGAAAGCACUACUAACAGCACU
165



GGAGGGUGUAGUGUUUCCUACUUUAUGGAUG


 


miR-143-1
GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGU
166



GCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUG



AAGCACUGUAGCUCAGGAAGAGAGAAGUUGUUC



UGCAGC


 


miR-143-2
CCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUG
167



GGAGUCUGAGAUGAAGCACUGUAGCUCAGG


 


miR-144-1
UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGU
168



AAGUUUGCGAUGAGACACUACAGUAUAGAUGAU



GUACUAGUCCGGGCACCCCC


 


miR-144-2
GGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCG
169



AUGAGACACUACAGUAUAGAUGAUGUACUAGUC


 


miR-145-1
CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAA
170



UCCCUUAGAUGCUAAGAUGGGGAUUCCUGGAAA



UACUGUUCUUGAGGUCAUGGUU


 


miR-145-2
CUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGA
171



UGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCU



UGAG


 


miR-146-1
CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUU
172



CCAUGGGUUGUGUCAGUGUCAGACCUCUGAAAU



UCAGUUCUUCAGCUGGGAUAUCUCUGUCAUCGU


 


miR-146-2
AGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCA
173



GUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU


 


miR-147
AAUCUAAAGACAACAUUUCUGCACACACACCAG
174



ACUAUGGAAGCCAGUGUGUGGAAAUGCUUCUGC



UAGAUU


 


miR-148a
GAGGCAAAGUUCUGAGACACUCCGACUCUGAGU
175


(miR-148)
AUGAUAGAAGUCAGUGCACUACAGAACUUUGUC



UC


 


miR-148b
CAAGCACGAUUAGCAUUUGAGGUGAAGUUCUGU
176



UAUACACUCAGGCUGUGGCUCUCUGAAAGUCAG



UGCAUCACAGAACUUUGUCUCGAAAGCUUUCUA


 


miR-148b-
AAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUU
177


small
AUACACUCAGGCUGUGGCUCUCUGAAAGUCAGU



GCAU


 


miR-149-1
GCCGGCGCCCGAGCUCUGGCUCCGUGUCUUCACU
178



CCCGUGCUUGUCCGAGGAGGGAGGGAGGGACGG



GGGCUGUGCUGGGGCAGCUGGA


 


miR-149-2
GCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUC
179



CGAGGAGGGAGGGAGGGAC


 


miR-150-1
CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCA
180



GUGCUGGGCUCAGACCCUGGUACAGGCCUGGGG



GACAGGGACCUGGGGAC


 


miR-150-2
CCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCU
181



CAGACCCUGGUACAGGCCUGGGGGACAGGG


 


miR-151
UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAU
182



GUCUCAUCCCCUACUAGACUGAAGCUCCUUGAG



GACAGG


 


miR-151-2
CCUGUCCUCAAGGAGCUUCAGUCUAGUAGGGGA
183



UGAGACAUACUAGACUGUGAGCUCCUCGAGGGC



AGG


 


miR-152-1
UGUCCCCCCCGGCCCAGGUUCUGUGAUACACUCC
184



GACUCGGGCUCUGGAGCAGUCAGUGCAUGACAG



AACUUGGGCCCGGAAGGACC


 


miR-152-2
GGCCCAGGUUCUGUGAUACACUCCGACUCGGGC
185



UCUGGAGCAGUCAGUGCAUGACAGAACUUGGGC



CCCGG


 


miR-153-1-1
CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGC
186



AGCUAGUAUUCUCACUCCAGUUGCAUAGUCACA



AAAGUGAUCAUUGGCAGGUGUGGC


 


miR-153-1-2
UCUCUCUCUCCCUCACAGCUGCCAGUGUCAUUGU
187



CACAAAAGUGAUCAUUGGCAGGUGUGGCUGCUG



CAUG


 


miR-153-2-1
AGCGGUGGCCAGUGUCAUUUUUGUGAUGUUGCA
188



GCUAGUAAUAUGAGCCCAGUUGCAUAGUCACAA



AAGUGAUCAUUGGAAACUGUG


 


miR-153-2-2
CAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAU
189



AUGAGCCCAGUUGCAUAGUCACAAAAGUGAUCA



UUG


 


miR-154-1
GUGGUACUUGAAGAUAGGUUAUCCGUGUUGCCU
190



UCGCUUUAUUUGUGACGAAUCAUACACGGUUGA



CCUAUUUUUCAGUACCAA


 


miR-154-2
GAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAU
191



UUGUGACGAAUCAUACACGGUUGACCUAUUUUU


 


miR-155
CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCC
192



UCCAACUGACUCCUACAUAUUAGCAUUAACAG


 


miR-156 =
CCUAACACUGUCUGGUAAAGAUGGCUCCCGGGU
193


miR-
GGGUUCUCUCGGCAGUAACCUUCAGGGAGCCCU


157 = overlap
GAAGACCAUGGAGGAC


miR-141


 


miR-158-
GCCGAGACCGAGUGCACAGGGCUCUGACCUAUG
194


small = miR-
AAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGC


192
UGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCU



CAAUGCCAGC


 


miR-159-1-
UCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAG
195


small
UGCCCACUGGUUCCAGUGGGGCUGCUGUUAUCU



GGGGCGAGGGCCA


 


miR-161-
AAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGG
196


small
UGACUGGUCUGGGCUACGCUAUGCUGCGGCGCU



CGGG


 


miR-163-1b-
CAUUGGCCUCCUAAGCCAGGGAUUGUGGGUUCG
197


small
AGUCCCACCCGGGGUAAAGAAAGGCCGAAUU


 


miR-163-3-
CCUAAGCCAGGGAUUGUGGGUUCGAGUCCCACC
198


small
UGGGGUAGAGGUGAAAGUUCCUUUUACGGAAUU



UUUU


 


miR-162
CAAUGUCAGCAGUGCCUUAGCAGCACGUAAAUA
199



UUGGCGUUAAGAUUCUAAAAUUAUCUCCAGUAU



UAACUGUGCUGCUGAAGUAAGGUUGACCAUACU



CUACAGUUG


 


miR-175-
GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUA
200


small = miR-
GAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUA


224
GUGACUACAAAGCCC


 


miR-177-
ACGCAAGUGUCCUAAGGUGAGCUCAGGGAGCAC
201


small
AGAAACCUCCAGUGGAACAGAAGGGCAAAAGCU



CAUU


 


miR-180-
CAUGUGUCACUUUCAGGUGGAGUUUCAAGAGUC
202


small
CCUUCCUGGUUCACCGUCUCCUUUGCUCUUCCAC



AAC


 


miR-181a
AGAAGGGCUAUCAGGCCAGCCUUCAGAGGACUC
203



CAAGGAACAUUCAACGCUGUCGGUGAGUUUGGG



AUUUGAAAAAACCACUGACCGUUGACUGUACCU



UGGGGUCCUUA


 


miR-181b-1
CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAA
204



UCAACAUUCAUUGCUGUCGGUGGGUUGAACUGU



GUGGACAAGCUCACUGAACAAUGAAUGCAACUG



UGGCCCCGCUU


 


miR-181b-2
CUGAUGGCUGCACUCAACAUUCAUUGCUGUCGG
205



UGGGUUUGAGUCUGAAUCAACUCACUGAUCAAU



GAAUGCAAACUGCGGACCAAACA


 


miR-181c
CGGAAAAUUUGCCAAGGGUUUGGGGGAACAUUC
206



AACCUGUCGGUGAGUUUGGGCAGCUCAGGCAAA



CCAUCGACCGUUGAGUGGACCCUGAGGCCUGGA



AUUGCCAUCCU


 


miR-182-as
GAGCUGCUUGCCUCCCCCCGUUUUUGGCAAUGG
207



UAGAACUCACACUGGUGAGGUAACAGGAUCCGG



UGGUUCUAGACUUGCCAACUAUGGGGCGAGGAC



UCAGCCGGCAC


 


miR-182
UUUUUGGCAAUGGUAGAACUCACACUGGUGAGG
208



UAACAGGAUCCGGUGGUUCUAGACUUGCCAACU



AUGG


 


miR-183
CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCA
209



CUGGUAGAAUUCACUGUGAACAGUCUCAGUCAG



UGAAUUACCGAAGGGCCAUAAACAGAGCAGAGA



CAGAUCCACGA


 


miR-184-1
CCAGUCACGUCCCCUUAUCACUUUUCCAGCCCAG
210



CUUUGUGACUGUAAGUGUUGGACGGAGAACUGA



UAAGGGUAGGUGAUUGA


 


miR-184-2
CCUUAUCACUUUUCCAGCCCAGCUUUGUGACUG
211



UAAGUGUUGGACGGAGAACUGAUAAGGGUAGG


 


miR-185-1
AGGGGGCGAGGGAUUGGAGAGAAAGGCAGUUCC
212



UGAUGGUCCCCUCCCCAGGGGCUGGCUUUCCUCU



GGUCCUUCCCUCCCA


 


miR-185-2
AGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUC
213



CCCUCCCCAGGGGCUGGCUUUCCUCUGGUCCUU


 


miR-186-1
UGCUUGUAACUUUCCAAAGAAUUCUCCUUUUGG
214



GCUUUCUGGUUUUAUUUUAAGCCCAAAGGUGAA



UUUUUUGGGAAGUUUGAGCU


 


miR-186-2
ACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUG
215



GUUUUAUUUUAAGCCCAAAGGUGAAUUUUUUGG



GAAGU


 


miR-187
GGUCGGGCUCACCAUGACACAGUGUGAGACUCG
216



GGCUACAACACAGGACCCGGGGCGCUGCUCUGA



CCCCUCGUGUCUUGUGUUGCAGCCGGAGGGACG



CAGGUCCGCA


 


miR-188-1
UGCUCCCUCUCUCACAUCCCUUGCAUGGUGGAG
217



GGUGAGCUUUCUGAAAACCCCUCCCACAUGCAG



GGUUUGCAGGAUGGCGAGCC


 


miR-188-2
UCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUU
218



UCUGAAAACCCCUCCCACAUGCAGGGUUUGCAG



GA


 


miR-189-1
CUGUCGAUUGGACCCGCCCUCCGGUGCCUACUGA
219



GCUGAUAUCAGUUCUCAUUUUACACACUGGCUC



AGUUCAGCAGGAACAGGAGUCGAGCCCUUGAGC



AA


 


miR-189-2
CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCA
220



UUUUACACACUGGCUCAGUUCAGCAGGAACAGG



AG


 


miR-190-1
UGCAGGCCUCUGUGUGAUAUGUUUGAUAUAUUA
221



GGUUGUUAUUUAAUCCAACUAUAUAUCAAACAU



AUUCCUACAGUGUCUUGCC


 


miR-190-2
CUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAU
222



UUAAUCCAACUAUAUAUCAAACAUAUUCCUACAG


 


miR-191-1
CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGC
223



AGCUGUUGUCUCCAGAGCAUUCCAGCUGCGCUU



GGAUUUCGUCCCCUGCUCUCCUGCCU


 


miR-191-2
AGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGU
224



CUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGU



CCCCUGCU


 


miR-192-2/3
CCGAGACCGAGUGCACAGGGCUCUGACCUAUGA
225



AUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGCU



GCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUC



AAUGCCAG


 


miR-192
GCCGAGACCGAGUGCACAGGGCUCUGACCUAUG
226



AAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGC



UGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCU



CAAUGCCAGC


 


miR-193-1
CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGG
227



GCGAGAUGAGGGUGUCGGAUCAACUGGCCUACA



AAGUCCCAGUUCUCGGCCCCCG


 


miR-193-2
GCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGG
228



AUCAACUGGCCUACAAAGUCCCAGU


 


miR-194-1
AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGU
229



GGACUGUGUACCAAUUUCCAGUGGAGAUGCUGU



UACUUUUGAUGGUUACCAA


 


miR-194-2
GUGUAACAGCAACUCCAUGUGGACUGUGUACCA
230



AUUUCCAGUGGAGAUGCUGUUACUUUUGAU


 


miR-195-1
AGCUUCCCUGGCUCUAGCAGCACAGAAAUAUUG
231



GCACAGGGAAGCGAGUCUGCCAAUAUUGGCUGU



GCUGCUCCAGGCAGGGUGGUG


 


miR-195-2
UAGCAGCACAGAAAUAUUGGCACAGGGAAGCGA
232



GUCUGCCAAUAUUGGCUGUGCUGCU


 


miR-196-1
CUAGAGCUUGAAUUGGAACUGCUGAGUGAAUUA
233



GGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGA



ACACAACAACAUUAAACCACCCGAUUCACGGCA



GUUACUGCUCC


 


miR-196a-1
GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGG
234



GUUUCUGAACACAACAACAUUAAACCACCCGAU



UCAC


 


miR-196a-2
UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUU
235


(miR-196-2)
UCAUGUUGUUGGGAUUGAGUUUUGAACUCGGCA



ACAAGAAACUGCCUGAGUUACAUCAGUCGGUUU



UCGUCGAGGGC


 


miR-196
GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGG
236



GUUUCUGAACACAACAACAUUAAACCACCCGAU



UCAC


 


miR-196b
ACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUU
237



GGGAUCCACCUUUCUCUCGACAGCACGACACUGC



CUUCAUUACUUCAGUUG


 


miR-197
GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUA
238



AGAGCUCUUCACCCUUCACCACCUUCUCCACCCA



GCAUGGCC


 


miR-197-2
GUGCAUGUGUAUGUAUGUGUGCAUGUGCAUGUG
239



UAUGUGUAUGAGUGCAUGCGUGUGUGC


 


miR-198
UCAUUGGUCCAGAGGGGAGAUAGGUUCCUGUGA
240



UUUUUCCUUCUUCUCUAUAGAAUAAAUGA


 


miR-199a-1
GCCAACCCAGUGUUCAGACUACCUGUUCAGGAG
241



GCUCUCAAUGUGUACAGUAGUCUGCACAUUGGU



UAGGC


 


miR-199a-2
AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCC
242



AGUGUUCAGACUACCUGUUCAGGACAAUGCCGU



UGUACAGUAGUCUGCACAUUGGUUAGACUGGGC



AAGGGAGAGCA


 


miR-199b
CCAGAGGACACCUCCACUCCGUCUACCCAGUGUU
243



UAGACUAUCUGUUCAGGACUCCCAAAUUGUACA



GUAGUCUGCACAUUGGUUAGGCUGGGCUGGGUU



AGACCCUCGG


 


miR-199s
GCCAACCCAGUGUUCAGACUACCUGUUCAGGAG
244



GCUCUCAAUGUGUACAGUAGUCUGCACAUUGGU



UAGGC


 


miR-200a
GCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGG
245



AGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGA



CGGC


 


miR-200b
CCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGC
246



AGCAUUGGAUGGAGUCAGGUCUCUAAUACUGCC



UGGUAAUGAUGACGGCGGAGCCCUGCACG


 


miR-200c
CCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGU
247



UGGGAGUCUCUAAUACUGCCGGGUAAUGAUGGA



GG


 


miR-202
GUUCCUUUUUCCUAUGCAUAUACUUCUUUGAGG
248



AUCUGGCCUAAAGAGGUAUAGGGCAUGGGAAGA



UGGAGC


 


miR-203
GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUU
249



CUUAACAGUUCAACAGUUCUGUAGCGCAAUUGU



GAAAUGUUUAGGACCACUAGACCCGGCGGGCGC



GGCGACAGCGA


 


miR-204
GGCUACAGUCUUUCUUCAUGUGACUCGUGGACU
250



UCCCUUUGUCAUCCUAUGCCUGAGAAUAUAUGA



AGGAGGCUGGGAAGGCAAAGGGACGUUCAAUUG



UCAUCACUGGC


 


miR-205
AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUG
251



UCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAA



CCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAU



GGAGCUGACA


 


miR-206-1
UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCC
252



AUAUGGAUUACUUUGCUAUGGAAUGUAAGGAAG



UGUGUGGUUUCGGCAAGUG


 


miR-206-2
AGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAU
253



UACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGU



UUU


 


miR-208
UGACGGGCGAGCUUUUGGCCCGGGUUAUACCUG
254



AUGCUCACGUAUAAGACGAGCAAAAAGCUUGUU



GGUCA


 


miR-210
ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCU
255



GCCCACCGCACACUGCGCUGCCCCAGACCCACUG



UGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCA



GCGCGACCC


 


miR-211
UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUU
256



GUCAUCCUUCGCCUAGGGCUCUGAGCAGGGCAG



GGACAGCAAAGGGGUGCUCAGUUGUCACUUCCC



ACAGCACGGAG


 


miR-212
CGGGGCACCCCGCCCGGACAGCGCGCCGGCACCU
257



UGGCUCUAGACUGCUUACUGCCCGGGCCGCCCUC



AGUAACAGUCUCCAGUCACGGCCACCGACGCCUG



GCCCCGCC


 


miR-213-2
CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAA
258



UCAACAUUCAUUGCUGUCGGUGGGUUGAACUGU



GUGGACAAGCUCACUGAACAAUGAAUGCAACUG



UGGCCCCGCUU


 


miR-213
GAGUUUUGAGGUUGCUUCAGUGAACAUUCAACG
259



CUGUCGGUGAGUUUGGAAUUAAAAUCAAAACCA



UCGACCGUUGAUUGUACCCUAUGGCUAACCAUC



AUCUACUCC


 


miR-214
GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCC
260



UGUCUACACUUGCUGUGCAGAACAUCCGCUCAC



CUGUACAGCAGGCACAGACAGGCAGUCACAUGA



CAACCCAGCCU


 


miR-215
AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCU
261



AUGAAUUGACAGACAAUAUAGCUGAGUUUGUCU



GUCAUUUCUUUAGGCCAAUAUUCUGUAUGACUG



UGCUACUUCAA


 


miR-216
GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCA
262



ACUGUGAGAUGUUCAUACAAUCCCUCACAGUGG



UCUCUGGGAUUAUGCUAAACAGAGCAAUUUCCU



AGCCCUCACGA


 


miR-217
AGUAUAAUUAUUACAUAGUUUUUGAUGUCGCAG
263



AUACUGCAUCAGGAACUGAUUGGAUAAGAAUCA



GUCACCAUCAGUUCCUAAUGCAUUGCCUUCAGC



AUCUAAACAAG


 


miR-218-1
GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUG
264



AUCUAACCAUGUGGUUGCGAGGUAUGAGUAAAA



CAUGGUUCCGUCAAGCACCAUGGAACGUCACGC



AGCUUUCUACA


 


miR-218-2
GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUG
265



AUCUAACCAUGUGGUGGAACGAUGGAAACGGAA



CAUGGUUCUGUCAAGCACCGCGGAAAGCACCGU



GCUCUCCUGCA


 


miR-219
CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACG
266



CAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUU



GAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACC



UCGAGCGGG


 


miR-219-1
CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACG
267



CAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUU



GAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACC



UCGAGCGGG


 


miR-219-2
ACUCAGGGGCUUCGCCACUGAUUGUCCAAACGC
268



AAUUCUUGUACGAGUCUGCGGCCAACCGAGAAU



UGUGGCUGGACAUCUGUGGCUGAGCUCCGGG


 


miR-220
GACAGUGUGGCAUUGUAGGGCUCCACACCGUAU
269



CUGACACUUUGGGCGAGGGCACCAUGCUGAAGG



UGUUCAUGAUGCGGUCUGGGAACUCCUCACGGA



UCUUACUGAUG


 


miR-221
UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAU
270



ACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAG



CUACAUUGUCUGCUGGGUUUCAGGCUACCUGGA



AACAUGUUCUC


 


miR-222
GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUC
271



AGUAGCCAGUGUAGAUCCUGUCUUUCGUAAUCA



GCAGCUACAUCUGGCUACUGGGUCUCUGAUGGC



AUCUUCUAGCU


 


miR-223
CCUGGCCUCCUGCAGUGCCACGCUCCGUGUAUUU
272



GACAAGCUGAGUUGGACACUCCAUGUGGUAGAG



UGUCAGUUUGUCAAAUACCCCAAGUGCGGCACA



UGCUUACCAG


 


miR-224
GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUA
273



GAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUA



GUGACUACAAAGCCC


 


miR-294-1
CAAUCUUCCUUUAUCAUGGUAUUGAUUUUUCAG
274


(chr16)
UGCUUCCCUUUUGUGUGAGAGAAGAUA


 


miR-296
AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGU
275



UGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCU



CCUGAAGGGCUCU


 


miR-299
AAGAAAUGGUUUACCGUCCCACAUACAUUUUGA
276



AUAUGUAUGUGGGAUGGUAAACCGCUUCUU


 


miR-301
ACUGCUAACGAAUGCUCUGACUUUAUUGCACUA
277



CUGUACUUUACAGCUAGCAGUGCAAUAGUAUUG



UCAAAGCAUCUGAAAGCAGG


 


miR-302a
CCACCACUUAAACGUGGAUGUACUUGCUUUGAA
278



ACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGA



UGG


 


miR-302b
GCUCCCUUCAACUUUAACAUGGAAGUGCUUUCU
279



GUGACUUUAAAAGUAAGUGCUUCCAUGUUUUAG



UAGGAGU


 


miR-302c
CCUUUGCUUUAACAUGGGGGUACCUGCUGUGUG
280



AAACAAAAGUAAGUGCUUCCAUGUUUCAGUGGA



GG


 


miR-302d
CCUCUACUUUAACAUGGAGGCACUUGCUGUGAC
281



AUGACAAAAAUAAGUGCUUCCAUGUUUGAGUGU



GG


 


miR-320
GCUUCGCUCCCCUCCGCCUUCUCUUCCCGGUUCU
282



UCCCGGAGUCGGGAAAAGCUGGGUUGAGAGGGC



GAAAAAGGAUGAGGU


 


miR-321
UUGGCCUCCUAAGCCAGGGAUUGUGGGUUCGAG
283



UCCCACCCGGGGUAAAGAAAGGCCGA


 


miR-323
UUGGUACUUGGAGAGAGGUGGUCCGUGGCGCGU
284



UCGCUUUAUUUAUGGCGCACAUUACACGGUCGA



CCUCUUUGCAGUAUCUAAUC


 


miR-324
CUGACUAUGCCUCCCCGCAUCCCCUAGGGCAUUG
285



GUGUAAAGCUGGAGACCCACUGCCCCAGGUGCU



GCUGGGGGUUGUAGUC


 


miR-325
AUACAGUGCUUGGUUCCUAGUAGGUGUCCAGUA
286



AGUGUUUGUGACAUAAUUUGUUUAUUGAGGACC



UCCUAUCAAUCAAGCACUGUGCUAGGCUCUGG


 


miR-326
CUCAUCUGUCUGUUGGGCUGGAGGCAGGGCCUU
287



UGUGAAGGCGGGUGGUGCUCAGAUCGCCUCUGG



GCCCUUCCUCCAGCCCCGAGGCGGAUUCA


 


miR-328
UGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAA
288



AGUGCAUACAGCCCCUGGCCCUCUCUGCCCUUCC



GUCCCCUG


 


miR-330
CUUUGGCGAUCACUGCCUCUCUGGGCCUGUGUC
289



UUAGGCUCUGCAAGAUCAACCGAGCAAAGCACA



CGGCCUGCAGAGAGGCAGCGCUCUGCCC


 


miR-331
GAGUUUGGUUUUGUUUGGGUUUGUUCUAGGUAU
290



GGUCCCAGGGAUCCCAGAUCAAACCAGGCCCCUG



GGCCUAUCCUAGAACCAACCUAAGCUC


 


miR-335
UGUUUUGAGCGGGGGUCAAGAGCAAUAACGAAA
291



AAUGUUUGUCAUAAACCGUUUUUCAUUAUUGCU



CCUGACCUCCUCUCAUUUGCUAUAUUCA


 


miR-337
GUAGUCAGUAGUUGGGGGGUGGGAACGGCUUCA
292



UACAGGAGUUGAUGCACAGUUAUCCAGCUCCUA



UAUGAUGCCUUUCUUCAUCCCCUUCAA


 


miR-338
UCUCCAACAAUAUCCUGGUGCUGAGUGAUGACU
293



CAGGCGACUCCAGCAUCAGUGAUUUUGUUGAAGA


 


miR-339
CGGGGCGGCCGCUCUCCCUGUCCUCCAGGAGCUC
294



ACGUGUGCCUGCCUGUGAGCGCCUCGACGACAG



AGCCGGCGCCUGCCCCAGUGUCUGCGC


 


miR-340
UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACU
295



GAUUGUCAUAUGUCGUUUGUGGGAUCCGUCUCA



GUUACUUUAUAGCCAUACCUGGUAUCUUA


 


miR-342
GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGU
296



GAUUGAGGGACAUGGUUAAUGGAAUUGUCUCAC



ACAGAAAUCGCACCCGUCACCUUGGCCUACUUA


 


miR-345
ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAG
297



GGCUCGUGAUGGCUGGUGGGCCCUGAACGAGGG



GUCUGGAGGCCUGGGUUUGAAUAUCGACAGC


 


miR-346
GUCUGUCUGCCCGCAUGCCUGCCUCUCUGUUGCU
298



CUGAAGGAGGCAGGGGCUGGGCCUGCAGCUGCC



UGGGCAGAGCGGCUCCUGC


 


miR-367
CCAUUACUGUUGCUAAUAUGCAACUCUGUUGAA
299



UAUAAAUUGGAAUUGCACUUUAGCAAUGGUGAU



GG


 


miR-368
AAAAGGUGGAUAUUCCUUCUAUGUUUAUGUUAU
300



UUAUGGUUAAACAUAGAGGAAAUUCCACGUUUU


 


miR-369
UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUU
301



AUUGACUUCGAAUAAUACAUGGUUGAUCUUUUC



UCAG


 


miR-370
AGACAGAGAAGCCAGGUCACGUCUCUGCAGUUA
302



CACAGCUCACGAGUGCCUGCUGGGGUGGAACCU



GGUCUGUCU


 


miR-371
GUGGCACUCAAACUGUGGGGGCACUUUCUGCUC
303



UCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUAC


 


miR-372
GUGGGCCUCAAAUGUGGAGCACUAUUCUGAUGU
304



CCAAGUGGAAAGUGCUGCGACAUUUGAGCGUCAC


 


miR-373
GGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUG
305



UCUGUACUGGGAAGUGCUUCGAUUUUGGGGUGU



CCC


 


miR-374
UACAUCGGCCAUUAUAAUACAACCUGAUAAGUG
306



UUAUAGCACUUAUCAGAUUGUAUUGUAAUUGUC



UGUGUA


 


miR-hes1
AUGGAGCUGCUCACCCUGUGGGCCUCAAAUGUG
307



GAGGAACUAUUCUGAUGUCCAAGUGGAAAGUGC



UGCGACAUUUGAGCGUCACCGGUGACGCCCAUA



UCA


 


miR-hes2
GCAUCCCCUCAGCCUGUGGCACUCAAACUGUGG
308



GGGCACUUUCUGCUCUCUGGUGAAAGUGCCGCC



AUCUUUUGAGUGUUACCGCUUGAGAAGACUCAA



CC


 


miR-hes3
CGAGGAGCUCAUACUGGGAUACUCAAAAUGGGG
309



GCGCUUUCCUUUUUGUCUGUUACUGGGAAGUGC



UUCGAUUUUGGGGUGUCCCUGUUUGAGUAGGGC



AUC





*An underlined sequence within a precursor sequence corresponds to a mature processed miR transcript (see Table 1b). Some precursor sequences have two underlined sequences denoting two different mature miRs that are derived from the same precursor. All sequences are human.













TABLE 1b







Human Mature microRNA Sequences.











Mature






miRNA
Mature miRNA Sequence
SEQ ID
Corresponding precursor


Name
(5′ to 3′)
NO.
microRNA(s); see Table 1a





let-7a
ugagguaguagguuguauaguu
310
let-7a-1; let-7a-2; let-7a-3;






let-7a-4


 


let-7b
ugagguaguagguugugugguu
311
let-7b


 


let-7c
ugagguaguagguuguaugguu
312
let-7c


 


let-7d
agagguaguagguugcauagu
313
let-7d; let-7d-v1


 


let-7e
ugagguaggagguuguauagu
314
let-7e


 


let-7f
ugagguaguagauuguauaguu
315
let-7f-1; let-7f-2-1;





let-7f-2-2


 


let-7g
ugagguaguaguuuguacagu
316
let-7g


 


let-7i
ugagguaguaguuugugcu
317
let-7i


 


miR-1
uggaauguaaagaaguaugua
318
miR-1b; miR-1b-1;





miR-1b-2


 


miR-7
uggaagacuagugauuuuguu
319
miR-7-1; miR-7-1a;





miR-7-2; miR-7-3


 


miR-9
ucuuugguuaucuagcuguauga
320
miR-9-1; miR-9-2;





miR-9-3


 


miR-9*
uaaagcuagauaaccgaaagu
321
miR-9-1; miR-9-2;





miR-9-3


 


miR-10a
uacccuguagauccgaauuugug
322
miR-10a


 


miR-10b
uacccuguagaaccgaauuugu
323
miR-10b


 


miR-15a
uagcagcacauaaugguuugug
324
miR-15a; miR-15a-2


 


miR-15b
uagcagcacaucaugguuuaca
325
miR-15b


 


miR-16
uagcagcacguaaauauuggcg
326
miR-16-1; miR-16-2;





miR-16-13


 


miR-17-
caaagugcuuacagugcagguagu
327
miR-17


5p


 


miR-17-
acugcagugaaggcacuugu
328
miR-17


3p


 


miR-18
uaaggugcaucuagugcagaua
329
miR-18; miR-18-13


 


miR-19a
ugugcaaaucuaugcaaaacuga
330
miR-19a; miR-19a-13


 


miR-19b
ugugcaaauccaugcaaaacuga
331
miR-19b-1; miR-19b-2


 


miR-20
uaaagugcuuauagugcaggua
332
miR-20 (miR-20a)


 


miR-21
uagcuuaucagacugauguuga
333
miR-21; miR-21-17


 


miR-22
aagcugccaguugaagaacugu
334
miR-22


 


miR-23a
aucacauugccagggauuucc
335
miR-23a


 


miR-23b
aucacauugccagggauuaccac
336
miR-23b


 


miR-24
uggcucaguucagcaggaacag
337
miR-24-1; miR-24-2;





miR-24-19; miR-24-9


 


miR-25
cauugcacuugucucggucuga
338
miR-25


 


miR-26a
uucaaguaauccaggauaggcu
339
miR-26a; miR-26a-1;





miR-26a-2


 


miR-26b
uucaaguaauucaggauaggu
340
miR-26b


 


miR-27a
uucacaguggcuaaguuccgcc
341
miR-27a


 


miR-27b
uucacaguggcuaaguucug
342
miR-27b-1; miR-27b-2


 


miR-28
aaggagcucacagucuauugag
343
miR-28


 


miR-29a
cuagcaccaucugaaaucgguu
344
miR-29a-2; miR-29a


 


miR-29b
uagcaccauuugaaaucagu
345
miR-29b-1; miR-29b-2


 


miR-29c
uagcaccauuugaaaucgguua
346
miR-29c


 


miR-30a-
uguaaacauccucgacuggaagc
347
miR-30a


5p


 


miR-30a-
cuuucagucggauguuugcagc
348
miR-30a


3p


 


miR-30b
uguaaacauccuacacucagc
349
miR-30b-1; miR-30b-2


 


miR-30c
uguaaacauccuacacucucagc
350
miR-30c


 


miR-30d
uguaaacauccccgacuggaag
351
miR-30d


 


miR-30e
uguaaacauccuugacugga
352
miR-30e


 


miR-31
ggcaagaugcuggcauagcug
353
miR-31


 


miR-32
uauugcacauuacuaaguugc
354
miR-32


 


miR-33
gugcauuguaguugcauug
355
miR-33; miR-33b


 


miR-34a
uggcagugucuuagcugguugu
356
miR-34a


 


miR-34b
aggcagugucauuagcugauug
357
miR-34b


 


miR-34c
aggcaguguaguuagcugauug
358
miR-34c


 


miR-92
uauugcacuugucccggccugu
359
miR-92-2; miR-92-1


 


miR-93
aaagugcuguucgugcagguag
360
miR-93-1; miR-93-2


 


miR-95
uucaacggguauuuauugagca
361
miR-95


 


miR-96
uuuggcacuagcacauuuuugc
362
miR-96


 


miR-98
ugagguaguaaguuguauuguu
363
miR-98


 


miR-99a
aacccguagauccgaucuugug
364
miR-99a


 


miR-99b
cacccguagaaccgaccuugcg
365
miR-99b


 


miR-100
uacaguacugugauaacugaag
366
miR-100


 


miR-101
uacaguacugugauaacugaag
367
miR-101-1; miR-101-2


 


miR-103
agcagcauuguacagggcuauga
368
miR-103-1


 


miR-105
ucaaaugcucagacuccugu
369
miR-105


 


miR-106-a
aaaagugcuuacagugcagguagc
370
miR-106-a


 


miR-106-b
uaaagugcugacagugcagau
371
miR-106-b


 


miR-107
agcagcauuguacagggcuauca
372
miR-107


 


miR-122a
uggagugugacaaugguguuugu
373
miR-122a-1; miR-122a-2


 


miR-124a
uuaaggcacgcggugaaugcca
374
miR-124a-1; miR-124a-2;





miR-124a-3


 


miR-125a
ucccugagacccuuuaaccugug
375
miR-125a-1; miR-125a-2


 


miR-125b
ucccugagacccuaacuuguga
376
miR-125b-1; miR-125b-2


 


miR-126*
cauuauuacuuuugguacgcg
377
miR-126-1; miR-126-2


 


miR-126
ucguaccgugaguaauaaugc
378
miR-126-1; miR-126-2


 


miR-127
ucggauccgucugagcuuggcu
379
miR-127-1; miR-127-2


 


miR-128a
ucacagugaaccggucucuuuu
380
miR-128; miR-128a


 


miR-128b
ucacagugaaccggucucuuuc
381
miR-128b


 


miR-129
cuuuuugcggucugggcuugc
382
miR-129-1; miR-129-2


 


miR-130a
cagugcaauguuaaaagggc
383
miR-130a


 


miR-130b
cagugcaaugaugaaagggcau
384
miR-130b


 


miR-132
uaacagucuacagccauggucg
385
miR-132-1


 


miR-133a
uugguccccuucaaccagcugu
386
miR-133a-1; miR-133a-2


 


miR-133b
uugguccccuucaaccagcua
387
miR-133b


 


miR-134
ugugacugguugaccagaggg
388
miR-134-1; miR-134-2


 


miR-135a
uauggcuuuuuauuccuauguga
389
miR-135a; miR-135a-2





(miR-135-2)


 


miR-135b
uauggcuuuucauuccuaugug
390
miR-135b


 


miR-136
acuccauuuguuuugaugaugga
391
miR-136-1; miR-136-2


 


miR-137
uauugcuuaagaauacgcguag
392
miR-137


 


miR-138
agcugguguugugaauc
393
miR-138-1; miR-138-2


 


miR-139
ucuacagugcacgugucu
394
miR-139


 


miR-140
agugguuuuacccuaugguag
395
miR-140; miR-140as;





miR-140s


 


miR-141
aacacugucugguaaagaugg
396
miR-141-1; miR-141-2


 


miR-142-
uguaguguuuccuacuuuaugga
397
miR-142


3p


 


miR-142-
cauaaaguagaaagcacuac
398
miR-142


5p


 


miR-143
ugagaugaagcacuguagcuca
399
miR-143-1


 


miR-144
uacaguauagaugauguacuag
400
miR-144-1; miR-144-2


 


miR-145
guccaguuuucccaggaaucccuu
401
miR-145-1; miR-145-2


 


miR-146
ugagaacugaauuccauggguu
402
miR-146-1; miR-146-2


 


miR-147
guguguggaaaugcuucugc
403
miR-147


 


miR-148a
ucagugcacuacagaacuuugu
404
miR-148a (miR-148)


 


miR-148b
ucagugcaucacagaacuuugu
405
miR-148b


 


miR-149
ucuggcuccgugucuucacucc
406
miR-149


 


miR-150
ucucccaacccuuguaccagug
407
miR-150-1; miR-150-2


 


miR-151
acuagacugaagcuccuugagg
408
miR-151


 


miR-152
ucagugcaugacagaacuugg
409
miR-152-1; miR-152-2


 


miR-153
uugcauagucacaaaaguga
410
miR-153-1-1; miR-153-1-





2; miR-153-2-1;





miR-153-2-2


 


miR-154
uagguuauccguguugccuucg
411
miR-154-1; miR-154-2


 


miR-154*
aaucauacacgguugaccuauu
412
miR-154-1; miR-154-2


 


miR-155
uuaaugcuaaucgugauagggg
413
miR-155


 


miR-181a
aacauucaacgcugucggugagu
414
miR-181a


 


miR-181b
aacauucauugcugucgguggguu
415
miR-181b-1; miR-181b-2


 


miR-181c
aacauucaaccugucggugagu
416
miR-181c


 


miR-182
uuuggcaaugguagaacucaca
417
miR-182; miR-182as


 


miR-182*
ugguucuagacuugccaacua
418
miR-182; miR-182as


 


miR-183
uauggcacugguagaauucacug
419
miR-183


 


miR-184
uggacggagaacugauaagggu
420
miR-184-1; miR-184-2


 


miR-185
uggagagaaaggcaguuc
421
miR-185-1; miR-185-2


 


miR-186
caaagaauucuccuuuugggcuu
422
miR-186-1; miR-186-2


 


miR-187
ucgugucuuguguugcagccg
423
miR-187


 


miR-188
caucccuugcaugguggagggu
424
miR-188


 


miR-189
gugccuacugagcugauaucagu
425
miR-189-1; miR-189-2


 


miR-190
ugauauguuugauauauuaggu
426
miR-190-1; miR-190-2


 


miR-191
caacggaaucccaaaagcagcu
427
miR-191-1; miR-191-2


 


miR-192
cugaccuaugaauugacagcc
428
miR-192


 


miR-193
aacuggccuacaaagucccag
429
miR-193-1; miR-193-2


 


miR-194
uguaacagcaacuccaugugga
430
miR-194-1; miR-194-2


 


miR-195
uagcagcacagaaauauuggc
431
miR-195-1; miR-195-2


 


miR-196a
uagguaguuucauguuguugg
432
miR-196a; miR-196a-2





(miR196-2)


 


miR-196b
uagguaguuuccuguuguugg
433
miR-196b


 


miR-197
uucaccaccuucuccacccagc
434
miR-197


 


miR-198
gguccagaggggagauagg
435
miR-198


 


miR-199a
cccaguguucagacuaccuguuc
436
miR-199a-1; miR-199a-2


 


miR-
uacaguagucugcacauugguu
437
miR-199a-1; miR-199a-2;


199a*


miR-199s; miR-199b


 


miR-199b
cccaguguuuagacuaucuguuc
438
miR-199b


 


miR-200a
uaacacugucugguaacgaugu
439
miR-200a


 


miR-200b
cucuaauacugccugguaaugaug
440
miR-200b


 


miR-200c
aauacugccggguaaugaugga
441
miR-200c


 


miR-202
agagguauagggcaugggaaga
442
miR-202


 


miR-203
gugaaauguuuaggaccacuag
443
miR-203


 


miR-204
uucccuuugucauccuaugccu
444
miR-204


 


miR-205
uccuucauuccaccggagucug
445
miR-205


 


miR-206
uggaauguaaggaagugugugg
446
miR-206-1; miR-206-2


 


miR-208
auaagacgagcaaaaagcuugu
447
miR-208


 


miR-210
cugugcgugugacagcggcug
448
miR-210


 


miR-211
uucccuuugucauccuucgccu
449
miR-211


 


miR-212
uaacagucuccagucacggcc
450
miR-212


 


miR-213
accaucgaccguugauuguacc
451
miR-213


 


miR-214
acagcaggcacagacaggcag
452
miR-214


 


miR-215
augaccuaugaauugacagac
453
miR-215


 


miR-216
uaaucucagcuggcaacugug
454
miR-216


 


miR-217
uacugcaucaggaacugauuggau
455
miR-217


 


miR-218
uugugcuugaucuaaccaugu
456
miR-218-1; miR-218-2


 


miR-219
ugauuguccaaacgcaauucu
457
miR-219; miR-219-1;





miR-219-2


 


miR-220
ccacaccguaucugacacuuu
458
miR-220


 


miR-221
agcuacauugucugcuggguuuc
459
miR-221


 


miR-222
agcuacaucuggcuacugggucuc
460
miR-222


 


miR-223
ugucaguuugucaaauacccc
461
miR-223


 


miR-224
caagucacuagugguuccguuua
462
miR-224


 


miR-296
agggcccccccucaauccugu
463
miR-296


 


miR-299
ugguuuaccgucccacauacau
464
miR-299


 


miR-301
cagugcaauaguauugucaaagc
465
miR-301


 


miR-302a
uaagugcuuccauguuuugguga
466
miR-302a


 


miR-
acuuuaacauggaagugcuuucu
467
miR-302b


302b*


 


miR-302b
uaagugcuuccauguuuuaguag
468
miR-302b


 


miR-
uuuaacauggggguaccugcug
469
miR-302c


302c*


 


miR-302c
uaagugcuuccauguuucagugg
470
miR-302c


 


miR-302d
uaagugcuuccauguuugagugu
471
miR-302d


 


miR-320
aaaagcuggguugagagggcgaa
472
miR-320


 


miR-321
uaagccagggauuguggguuc
473
miR-321


 


miR-323
gcacauuacacggucgaccucu
474
miR-323


 


miR-324-
cgcauccccuagggcauuggugu
475
miR-324


5p


 


miR-324-
ccacugccccaggugcugcugg
476
miR-324


3p


 


miR-325
ccuaguagguguccaguaagu
477
miR-325


 


miR-326
ccucugggcccuuccuccag
478
miR-326


 


miR-328
cuggcccucucugcccuuccgu
479
miR-328


 


miR-330
gcaaagcacacggccugcagaga
480
miR-330


 


miR-331
gccccugggccuauccuagaa
481
miR-331


 


miR-335
ucaagagcaauaacgaaaaaugu
482
miR-335


 


miR-337
uccagcuccuauaugaugccuuu
483
miR-337


 


miR-338
uccagcaucagugauuuuguuga
484
miR-338


 


miR-339
ucccuguccuccaggagcuca
485
miR-339


 


miR-340
uccgucucaguuacuuuauagcc
486
miR-340


 


miR-342
ucucacacagaaaucgcacccguc
487
miR-342


 


miR-345
ugcugacuccuaguccagggc
488
miR-345


 


miR-346
ugucugcccgcaugccugccucu
489
miR-346


 


miR-367
aauugcacuuuagcaaugguga
490
miR-367


 


miR-368
acauagaggaaauuccacguuu
491
miR-368


 


miR-369
aauaauacaugguugaucuuu
492
miR-369


 


miR-370
gccugcugggguggaaccugg
493
miR-370


 


miR-371
gugccgccaucuuuugagugu
494
miR-371


 


miR-372
aaagugcugcgacauuugagcgu
495
miR-372


 


miR-373*
acucaaaaugggggcgcuuucc
496
miR-373


 


miR-373
gaagugcuucgauuuuggggugu
497
miR-373


 


miR-374
uuauaauacaaccugauaagug
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miR-374









The present invention encompasses methods of diagnosing whether a subject has, or is at risk for developing, a solid cancer, comprising measuring the level of at least one miR gene product in a test sample from the subject and comparing the level of the miR gene product in the test sample to the level of a corresponding miR gene product in a control sample. As used herein, a “subject” can be any mammal that has, or is suspected of having, a solid cancer. In a preferred embodiment, the subject is a human who has, or is suspected of having, a solid cancer.
In one embodiment, the at least one miR gene product measured in the test sample is selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof. In a particular embodiment, the miR gene product is miR-21, miR-191 or miR-17-5p. In another embodiment, the miR gene product is not miR-15a or miR-16-1. In an additional embodiment, the miR gene product is not miR 159-1 or miR-192. In an additional embodiment, the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the miR gene product is not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In another embodiment, the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let 7a, let-7f, or let-7d. In yet another embodiment, the miR gene product is not miR-15a, miR-16-1, miR-182, miR-181, miR-30, miR-15a, miR-16-1, miR-15b, miR-16-2, miR-195, miR-34, miR-153, miR-21, miR-217, miR-205, miR-204, miR-211, miR-143, miR-96, miR-103, miR-107, miR-129, miR-9, miR-137, miR-217, miR-186.
The solid cancer can be any cancer that arises from organs and solid tissues. Such cancers are typically associated with the formation and/or presence of tumor masses and can be carcinomas, sarcomas and lymphomas. Specific examples of solid cancers to be diagnosed by the methods of the invention include, but are not limited to, colon cancer, rectal cancer, stomach (gastric) cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, bronchial cancer, testicular cancer, ovarian cancer, uterine cancer, penile cancer, melanoma and other skin cancers, liver cancer, esophogeal cancer, cancers of the oral cavity and pharynx (e.g., tongue cancer, mouth cancer), cancers of the digestive system (e.g., intestinal cancer, gall bladder cancer), bone and joint cancers, cancers of the endocrine system (e.g., thyroid cancer), brain cancer, eye cancer, cancers of the urinary system (e.g., kidney cancer, urinary bladder cancer), Hodgkin disease and non-Hodgkin lymphoma. In particular embodiments, the solid cancer is not one or more of breast cancer, lung cancer, prostate cancer, pancreatic cancer or gastrointestinal cancer.
In one embodiment, the solid cancer is breast cancer or lung cancer and the at least one miR gene product measured in the test sample is selected from the group consisting of miR-210, miR-213 and a combination thereof.
In a further embodiment, the solid cancer is colon cancer, stomach cancer, prostate cancer or pancreas cancer and the at least one miR gene product measured in the test sample is miR-218-2.
In a certain embodiment of the invention, the solid cancer is breast cancer and the at least one miR gene product measured in the test sample is selected from the group consisting of miR-125b-1, miR-125b-2, miR-145, miR-21 and combinations thereof. In a related embodiment, the solid cancer is breast cancer and the at least one miR gene product in the test sample is selected from the group consisting of miR-21, miR-29b-2, miR-146, miR-125b-2, miR-125b-1, miR-10b, miR-145, miR-181a, miR-140, miR-213, miR-29a prec, miR-181b-1, miR-199b, miR-29b-1, miR-130a, miR-155, let-7a-2, miR-205, miR-29c, miR-224, miR-100, miR-31, miR-30c, miR-17-5p, miR-210, miR-122a, miR-16-2 and combinations thereof. In a related embodiment, the solid cancer is breast cancer and the at least one miR gene product is not miR-15a or miR-16-1. In a further embodiment, the solid cancer is breast cancer and the at least one miR gene product is not miR-145, miR-21, miR-155, miR-10b, miR-125b-1, miR-125b-2, let7a-2, let7a-3, let-7d, miR-122a, miR-191, miR-206, miR-210, let-71, miR-009-1 (miR131-1), miR-34 (miR-170), miR-102 (miR-29b), miR-123 (miR-126), miR-140-as, miR-125a, miR-194, miR-204, miR-213, let-7f-2, miR-101, miR-128b, miR-136, miR-143, miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-103-1, or miR-30c. In another embodiment, the solid cancer is breast cancer and the miR gene product is not miR-21, miR-125b-1, let-7a-2, let-71, miR-100, let-7g, miR-31, miR-32a-1, miR-33b, miR-34a-2, miR-101-1, miR-135-1, miR-142 as, miR-142s, miR-144, miR-301, miR-29c, miR-30c, miR-106a, or miR-29b-1. In yet another embodiment, the solid cancer is breast cancer and the miR gene product is not miR-159-1 or miR-192. In an additional embodiment, the solid cancer is breast cancer and the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is breast cancer and the miR gene product is not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is breast cancer and the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is breast cancer and the miR gene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.
In another embodiment, the solid cancer is colon cancer and the at least one miR gene product in the test sample is selected from the group consisting of miR-24-1, miR-29b-2, miR-20a, miR-10a, miR-32, miR-203, miR-106a, miR-17-5p, miR-30c, miR-223, miR-126*, miR-128b, miR-21, miR-24-2, miR-99b prec, miR-155, miR-213, miR-150, miR-107, miR-191, miR-221, miR-9-3 and combinations thereof. In another embodiment, the solid cancer is colon cancer and the miR gene product is not miR 159-1 or miR-192. In an additional embodiment, the solid cancer is colon cancer and the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is colon cancer and the miR gene product is not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is colon cancer and the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is colon cancer and the miR gene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.
In yet another embodiment, the solid cancer is lung cancer and the miR gene product in the test sample is selected from the group consisting of miR-21, miR-205, miR-200b, miR-9-1, miR-210, miR-148, miR-141, miR-132, miR-215, miR-128b, let-7g, miR-16-2, miR-129-1/2 prec, miR-126*, miR-142-as, miR-30d, miR-30a-5p, miR-7-2, miR-199a-1, miR-127, miR-34a prec, miR-34a, miR-136, miR-202, miR-196-2, miR-199a-2, let-7a-2, miR-124a-1, miR-149, miR-17-5p, miR-196-1 prec, miR-10a, miR-99b prec, miR-196-1, miR-199b, miR-191, miR-195, miR-155 and combinations thereof. In a related embodiment, the solid cancer is lung cancer and the at least one miR gene product is not miR-15a or miR-16-1. In a further embodiment, the solid cancer is lung cancer and the at least one miR gene product is not miR-21, miR-191, miR-126*, miR-210, miR-155, miR-143, miR-205, miR-126, miR-30a-5p, miR-140, miR-214, miR-218-2, miR-145, miR-106a, miR-192, miR-203, miR-150, miR-220, miR-192, miR-224, miR-24-2, miR-212, miR-9, miR-17, miR-124a-1, miR-95, miR-198, miR-216, miR-219-1; miR-197, miR-125a, miR-26a-1, miR-146, miR-199b, let7a-2, miR-27b, miR-32, miR-29b-2, miR-33, miR-181c, miR-101-1, miR-124a-3, miR-125b-1 or let7f-1. In another embodiment, the solid cancer is lung cancer and the at least one miR gene product is not miR-21, miR-182, miR-181, miR-30, miR-15a, miR-143, miR-205, miR-96, miR-103, miR-107, miR-129, miR-137, miR-186, miR-15b, miR-16-2, miR-195, miR-34, miR-153, miR-217, miR-204, miR-211, miR-9, miR-217, let-7a-2 or miR-32. In a further embodiment, the solid cancer is lung cancer and the miR gene product is not let-7c, let-7g, miR-7-3, miR-210, miR-31, miR-34a-1, miR-a-2, miR-99a, miR-100, miR-125b-2, miR-132, miR-135-1, miR-195, miR-34, miR-123, miR-203. In another embodiment, the solid cancer is lung cancer and the miR gene product is not miR 159-1 or miR-192. In an additional embodiment, the solid cancer is lung cancer and the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is lung cancer and the miR gene product is not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is lung cancer and the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is lung cancer and the miR gene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.
In a further embodiment, the solid cancer is pancreatic cancer and the at least one miR gene product measured in the test sample is selected from the group consisting of miR-103-1, miR-103-2, miR-155, miR-204 and combinations thereof. In a related embodiment, the solid cancer is pancreatic cancer and the miR gene product in the test sample is selected from the group consisting of miR-103-2, miR-103-1, miR-24-2, miR-107, miR-100, miR-125b-2, miR-125b-1, miR-24-1, miR-191, miR-23a, miR-26a-1, miR-125a, miR-130a, miR-26b, miR-145, miR-221, miR-126*, miR-16-2, miR-146, miR-214, miR-99b, miR-128b, miR-155, miR-29b-2, miR-29a, miR-25, miR-16-1, miR-99a, miR-224, miR-30d, miR-92-2, miR-199a-1, miR-223, miR-29c, miR-30b, miR-129-1/2, miR-197, miR-17-5p, miR-30c, miR-7-1, miR-93-1, miR-140, miR-30a-5p, miR-132, miR-181b-1, miR-152 prec, miR-23b, miR-20a, miR-222, miR-27a, miR-92-1, miR-21, miR-129-1/2 prec, miR-150, miR-32, miR-106a, miR-29b-1 and combinations thereof. In one embodiment, the solid cancer is pancreatic cancer and the miR gene product is not miR-15a or miR-16-1. In another embodiment, the solid cancer is pancreatic cancer and the miR gene product is not miR 159-1 or miR-192. In an additional embodiment, the solid cancer is pancreatic cancer and the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is pancreatic cancer and the miR gene product is not miR-21, miR-301, miR-142 as, miR-1425, miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is pancreatic cancer and the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is pancreatic cancer and the miR gene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.
In another embodiment, the solid cancer is prostate cancer and the miR gene product in the test sample is selected from the group consisting of let-7d, miR-128a prec, miR-195, miR-203, let-7a-2 prec, miR-34a, miR-20a, miR-218-2, miR-29a, miR-25, miR-95, miR-197, miR-135-2, miR-187, miR-196-1, miR-148, miR-191, miR-21, let-71, miR-198, miR-199a-2, miR-30c, miR-17-5p, miR-92-2, miR-146, miR-181b-1 prec, miR-32, miR-206, miR-184 prec, miR-29a prec, miR-29b-2, miR-149, miR-181b-1, miR-196-1 prec, miR-93-1, miR-223, miR-16-1, miR-101-1, miR-124a-1, miR-26a-1, miR-214, miR-27a, miR-24-1, miR-106a, miR-199a-1 and combinations thereof. In a related embodiment, the solid cancer is prostate cancer and the miR gene product is not miR-15a or miR-16-1. In another embodiment, the solid cancer is prostate cancer and the miR gene product is not miR 159-1 or miR-192. In an additional embodiment, the solid cancer is prostate cancer and the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is prostate cancer and the miR gene product is not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is prostate cancer and the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is prostate cancer and the miR gene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.
In yet another embodiment, the solid cancer is stomach cancer and the miR gene product in the test sample is selected from the group consisting of miR-223, miR-21, miR-218-2, miR-103-2, miR-92-2, miR-25, miR-136, miR-191, miR-221, miR-125b-2, miR-103-1, miR-214, miR-222, miR-212 prec, miR-125b-1, miR-100, miR-107, miR-92-1, miR-96, miR-192, miR-23a, miR-215, miR-7-2, miR-138-2, miR-24-1, miR-99b, miR-33b, miR-24-2 and combinations thereof. In a related embodiment, the solid cancer is stomach cancer and the miR gene product is not miR-15a or miR-16-1. In another embodiment, the solid cancer is stomach cancer and the miR gene product is not miR 159-1 or miR-192. In an additional embodiment, the solid cancer is stomach cancer and the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100; miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is stomach cancer and the miR gene product is not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is stomach cancer and the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is stomach cancer and the miR gene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.
The level of at least one miR gene product can be measured in a biological sample (e.g., cells, tissues) obtained from the subject. For example, a tissue sample (e.g., from a tumor) can be removed from a subject suspected of having a solid cancer by conventional biopsy techniques. In another embodiment, a blood sample can be removed from the subject, and blood cells (e.g., white blood cells) can be isolated for DNA extraction by standard techniques. The blood or tissue sample is preferably obtained from the subject prior to initiation of radiotherapy, chemotherapy or other therapeutic treatment. A corresponding control tissue or blood sample can be obtained from unaffected tissues of the subject, from a normal human individual or population of normal individuals, or from cultured cells corresponding to the majority of cells in the subject's sample. The control tissue or blood sample is then processed along with the sample from the subject, so that the levels of miR gene product produced from a given miR gene in cells from the subject's sample can be compared to the corresponding miR gene product levels from cells of the control sample. A reference miR expression standard for the biological sample can also be used as a control.
An alteration (e.g., an increase or decrease) in the level of a miR gene product in the sample obtained from the subject, relative to the level of a corresponding miR gene product in a control sample, is indicative of the presence of a solid cancer in the subject. In one embodiment, the level of the at least one miR gene product in the test sample is greater than the level of the corresponding miR gene product in the control sample (i.e., expression of the miR gene product is “up-regulated”). As used herein, expression of a miR gene product is “up-regulated” when the amount of miR gene product in a cell or tissue sample from a subject is greater than the amount of the same gene product in a control cell or tissue sample. In another embodiment, the level of the at least one miR gene product in the test sample is less than the level of the corresponding miR gene product in the control sample (i.e., expression of the miR gene product is “down-regulated”). As used herein, expression of a miR gene is “down-regulated” when the amount of miR gene product produced from that gene in a cell or tissue sample from a subject is less than the amount produced from the same gene in a control cell or tissue sample. The relative miR gene expression in the control and normal samples can be determined with respect to one or more RNA expression standards. The standards can comprise, for example, a zero miR gene expression level, the miR gene expression level in a standard cell line, the miR gene expression level in unaffected tissues of the subject, or the average level of miR gene expression previously obtained for a population of normal human controls.
The level of a miR gene product in a sample can be measured using any technique that is suitable for detecting RNA expression levels in a biological sample. Suitable techniques (e.g., Northern blot analysis, RT-PCR, in situ hybridization) for determining RNA expression levels in a biological sample (e.g., cells, tissues) are well known to those of skill in the art. In a particular embodiment, the level of at least one miR gene product is detected using Northern blot analysis. For example, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, the entire disclosure of which is incorporated by reference.
Suitable probes for Northern blot hybridization of a given miR gene product can be produced from the nucleic acid sequences provided in Table 1a and Table 1b and include, but are not limited to, probes having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or complete complementarity to a miR gene product of interest. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11, the disclosures of which are incorporated herein by reference.
For example, the nucleic acid probe can be labeled with, e.g., a radionuclide, such as 3H, 32P, 33P, 14C, or 35S; a heavy metal; a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin or an antibody); a fluorescent molecule; a chemiluminescent molecule; an enzyme or the like.
Probes can be labeled to high specific activity by either the nick translation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 or by the random priming method of Fienberg et al. (1983), Anal. Biochem. 132:6-13, the entire disclosures of which are incorporated herein by reference. The latter is the method of choice for synthesizing 32P-labeled probes of high specific activity from single-stranded DNA or from RNA templates. For example, by replacing preexisting nucleotides with highly radioactive nucleotides according to the nick translation method, it is possible to prepare 32P-labeled nucleic acid probes with a specific activity well in excess of 108 cpm/microgram. Autoradiographic detection of hybridization can then be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of miR gene transcript levels. Using another approach, miR gene transcript levels can be quantified by computerized imaging systems, such as the Molecular Dynamics 400-B 2D Phosphorimager available from Amersham Biosciences, Piscataway, N.J.
Where radionuclide labeling of DNA or RNA probes is not practical, the random-primer method can be used to incorporate an analogue, for example, the dTTP analogue 5-(N—(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate, into the probe molecule. The biotinylated probe oligonucleotide can be detected by reaction with biotin-binding proteins, such as avidin, streptavidin, and antibodies (e.g., anti-biotin antibodies) coupled to fluorescent dyes or enzymes that produce color reactions.
In addition to Northern and other RNA hybridization techniques, determining the levels of RNA transcripts can be accomplished using the technique of in situ hybridization. This technique requires fewer cells than the Northern blotting technique, and involves depositing whole cells onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labeled nucleic acid (e.g., cDNA or RNA) probes. This technique is particularly well-suited for analyzing tissue biopsy samples from subjects. The practice of the in situ hybridization technique is described in more detail in U.S. Pat. No. 5,427,916, the entire disclosure of which is incorporated herein by reference. Suitable probes for in situ hybridization of a given miR gene product can be produced from the nucleic acid sequences provided in Table 1a and Table 1b, and include, but are not limited to, probes having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or complete complementarity to a miR gene product of interest, as described above.
The relative number of miR gene transcripts in cells can also be determined by reverse transcription of miR gene transcripts, followed by amplification of the reverse-transcribed transcripts by polymerase chain reaction (RT-PCR). The levels of miR gene transcripts can be quantified in comparison with an internal standard, for example, the level of mRNA from a “housekeeping” gene present in the same sample. A suitable “housekeeping” gene for use as an internal standard includes, e.g., myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods for performing quantitative and semi-quantitative RT-PCR, and variations thereof, are well known to those of skill in the art.
In some instances, it may be desirable to simultaneously determine the expression level of a plurality of different miR gene products in a sample. In other instances, it may be desirable to determine the expression level of the transcripts of all known miR genes correlated with a cancer. Assessing cancer-specific expression levels for hundreds of miR genes or gene products is time consuming and requires a large amount of total RNA (e.g., at least 20 μg for each Northern blot) and autoradiographic techniques that require radioactive isotopes.
To overcome these limitations, an oligolibrary, in microchip format (i.e., a microarray), may be constructed containing a set of oligonucleotide (e.g., oligodeoxynucleotides) probes that are specific for a set of miR genes. Using such a microarray, the expression level of multiple microRNAs in a biological sample can be determined by reverse transcribing the RNAs to generate a set of target oligodeoxynucleotides, and hybridizing them to probe the oligonucleotides on the microarray to generate a hybridization, or expression, profile. The hybridization profile of the test sample can then be compared to that of a control sample to determine which microRNAs have an altered expression level in solid cancer cells. As used herein, “probe oligonucleotide” or “probe oligodeoxynucleotide” refers to an oligonucleotide that is capable of hybridizing to a target oligonucleotide. “Target oligonucleotide” or “target oligodeoxynucleotide” refers to a molecule to be detected (e.g., via hybridization). By “miR-specific probe oligonucleotide” or “probe oligonucleotide specific for a miR” is meant a probe oligonucleotide that has a sequence selected to hybridize to a specific miR gene product, or to a reverse transcript of the specific miR gene product.
An “expression profile” or “hybridization profile” of a particular sample is essentially a fingerprint of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. That is, normal tissue may be distinguished from cancerous (e.g., tumor) tissue, and within cancerous tissue, different prognosis states (for example, good or poor long term survival prospects) may be determined. By comparing expression profiles of solid cancer tissue in different states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. The identification of sequences that are differentially expressed in solid cancer tissue, as well as differential expression resulting in different prognostic outcomes, allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated (e.g., to determine whether a chemotherapeutic drug acts to improve the long-term prognosis in a particular patient). Similarly, diagnosis may be done or confirmed by comparing patient samples with known expression profiles. Furthermore, these gene expression profiles (or individual genes) allow screening of drug candidates that suppress the solid cancer expression profile or convert a poor prognosis profile to a better prognosis profile.
Accordingly, the invention provides methods of diagnosing whether a subject has, or is at risk for developing, a solid cancer, comprising reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the test sample, and comparing the test sample hybridization profile to a hybridization profile generated from a control sample or reference standard, wherein an alteration in the signal of at least one miRNA is indicative of the subject either having, or being at risk for developing, a solid cancer. In one embodiment, the microarray comprises miRNA-specific probe oligonucleotides for a substantial portion of all known human miRNAs. In a particular embodiment, the microarray comprises miRNA-specific probe oligonucleotides for one or more miRNAs selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
The microarray can be prepared from gene-specific oligonucleotide probes generated from known miRNA sequences. The array may contain two different oligonucleotide probes for each miRNA, one containing the active, mature sequence and the other being specific for the precursor of the miRNA. The array may also contain controls, such as one or more mouse sequences differing from human orthologs by only a few bases, which can serve as controls for hybridization stringency conditions. tRNAs or other RNAs (e.g., rRNAs, mRNAs) from both species may also be printed on the microchip, providing an internal, relatively stable, positive control for specific hybridization. One or more appropriate controls for non-specific hybridization may also be included on the microchip. For this purpose, sequences are selected based upon the absence of any homology with any known miRNAs.
The microarray may be fabricated using techniques known in the art. For example, probe oligonucleotides of an appropriate length, e.g., 40 nucleotides, are 5′-amine modified at position C6 and printed using commercially available microarray systems, e.g., the GeneMachine OmniGridTM 100 Microarrayer and Amersham CodeLinkTM activated slides. Labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcribing the target RNA with labeled primer. Following first strand synthesis, the RNA/DNA hybrids are denatured to degrade the RNA templates. The labeled target cDNAs thus prepared are then hybridized to the microarray chip under hybridizing conditions, e.g., 6×SSPE/30% formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT (Tris HCl/NaCl/Tween 20) at 37° C. for 40 minutes. At positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample, hybridization occurs. The labeled target cDNA marks the exact position on the array where binding occurs, allowing automatic detection and quantification. The output consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary miRs, in the patient sample. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of the biotin-containing transcripts using, e.g., Streptavidin-Alexa647 conjugate, and scanned utilizing conventional scanning methods. Image intensities of each spot on the array are proportional to the abundance of the corresponding miR in the patient sample.
The use of the array has several advantages for miRNA expression detection. First, the global expression of several hundred genes can be identified in the same sample at one time point. Second, through careful design of the oligonucleotide probes, expression of both mature and precursor molecules can be identified. Third, in comparison with Northern blot analysis, the chip requires a small amount of RNA, and provides reproducible results using 2.5 μg of total RNA. The relatively limited number of miRNAs (a few hundred per species) allows the construction of a common microarray for several species, with distinct oligonucleotide probes for each. Such a tool would allow for analysis of trans-species expression for each known miR under various conditions.
In addition to use for quantitative expression level assays of specific miRs, a microchip containing miRNA-specific probe oligonucleotides corresponding to a substantial portion of the miRNome, preferably the entire miRNome, may be employed to carry out miR gene expression profiling, for analysis of miR expression patterns. Distinct miR signatures can be associated with established disease markers, or directly with a disease state.
According to the expression profiling methods described herein, total RNA from a sample from a subject suspected of having a cancer (e.g., a solid cancer) is quantitatively reverse transcribed to provide a set of labeled target oligodeoxynucleotides complementary, to the RNA in the sample. The target oligodeoxynucleotides are then hybridized to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the sample. The result is a hybridization profile for the sample representing the expression pattern of miRNA in the sample. The hybridization profile comprises the signal from the binding of the target oligodeoxynucleotides from the sample to the miRNA-specific probe oligonucleotides in the microarray. The profile may be recorded as the presence or absence of binding (signal vs. zero signal). More preferably, the profile recorded includes the intensity of the signal from each hybridization. The profile is compared to the hybridization profile generated from a normal, i.e., noncancerous, control sample. An alteration in the signal is indicative of the presence of, or propensity to develop, cancer in the subject.
Other techniques for measuring miR gene expression are also within the skill in the art, and include various techniques for measuring rates of RNA transcription and degradation.
The invention also provides methods of determining the prognosis of a subject with a solid cancer, comprising measuring the level of at least one miR gene product, which is associated with a particular prognosis in a solid cancer (e.g., a good or positive prognosis, a poor or adverse prognosis), in a test sample from the subject. According to these methods, an alteration in the level of a miR gene product that is associated with a particular prognosis in the test sample, as compared to the level of a corresponding miR gene product in a control sample, is indicative of the subject having a solid cancer with a particular prognosis. In one embodiment, the miR gene product is associated with an adverse (i.e., poor) prognosis. Examples of an adverse prognosis include, but are not limited to, low survival rate and rapid disease progression. In certain embodiments, the level of the at least one miR gene product is measured by reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to a microarray that comprises miRNA-specific probe oligonucleotides to provide a hybridization profile for the test sample, and comparing the test sample hybridization profile to a hybridization profile generated from a control sample.
Without wishing to be bound by any one theory, it is believed that alterations in the level of one or more miR gene products in cells can result in the deregulation of one or more intended targets for these miRs, which can lead to the formation of solid cancers. Therefore, altering the level of the miR gene product (e.g., by decreasing the level of a miR gene product that is up-regulated in solid cancer cells, by increasing the level of a miR gene product that is down-regulated in solid cancer cells) may successfully treat the solid cancer.
Accordingly, the present invention encompasses methods of inhibiting tumorigenesis in a subject who has, or is suspected of having, a solid cancer wherein at least one miR gene product is deregulated (e.g., down-regulated, up-regulated) in the cancer cells of the subject. When the at least one isolated miR gene product is down-regulated in the cancer cells (e.g., miR-145, miR-155, miR-218-2), the method comprises administering an effective amount of the at least one isolated miR gene product, or an isolated variant or biologically-active fragment thereof, such that proliferation of cancer cells in the subject is inhibited. In one embodiment, the isolated miR gene product that is administered is not miR-15a or miR-16-1. In another embodiment, the miR gene product is not miR 159-1 or miR-192. In an additional embodiment, the miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182 as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142 as, miR-105-1, or miR-175. In a further embodiment, the miR gene product is not miR-21, miR-301, miR-142 as, miR-142s, miR-194, miR-215, or miR-32. In another embodiment, the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment, the miR gene product is not miR-30, miR-15b, miR-16-2, miR-217, miR-205, miR-204, miR-103, miR-107, miR-9, and miR-137. In a further embodiment, the miR gene product is not miR-145, miR-21, miR-155, miR-10b, miR-125b-1, miR-125b-2, let7a-2, let7a-3, let-7d, miR-122a, miR-191, miR-206, miR-210, let-71, miR-009-1 (miR131-1), miR-34 (miR-170), miR-102 (miR-29b), miR-123 (miR-126), miR-140-as, miR-125a, miR-194, miR-204, miR-213, let-7f-2, miR-101, miR-128b, miR-136, miR-143, miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-103-1, or miR-30c. In another embodiment, the miR gene product is not miR-21, miR-125b-1, let-7a-2, let-71, miR-100, let-7g, miR-31, miR-32a-1, miR-33b, miR-34a-2, miR-101-1, miR-135-1, miR-142 as, miR-142s, miR-144, miR-301, miR-29c, miR-30c, miR-106a, or miR-29b-1.
For example, when a miR gene product is down-regulated in a cancer cell in a subject, administering an effective amount of an isolated miR gene product to the subject can inhibit proliferation of the cancer cell. The isolated miR gene product that is administered to the subject can be identical to the endogenous wild-type miR gene product (e.g., a miR gene product shown in Table 1a or Table 1b) that is down-regulated in the cancer cell or it can be a variant or biologically-active fragment thereof. As defined herein, a “variant” of a miR gene product refers to a miRNA that has less than 100% identity to a corresponding wild-type miR gene product and possesses one or more biological activities of the corresponding wild-type miR gene product. Examples of such biological activities include, but are not limited to, inhibition of expression of a target RNA molecule (e.g., inhibiting translation of a target RNA molecule, modulating the stability of a target RNA molecule, inhibiting processing of a target RNA molecule) and inhibition of a cellular process associated with solid cancer (e.g., cell differentiation, cell growth, cell death). These variants include species variants and variants that are the consequence of one or more mutations (e.g., a substitution, a deletion, an insertion) in a miR gene. In certain embodiments, the variant is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a corresponding wild-type miR gene product.
As defined herein, a “biologically-active fragment” of a miR gene product refers to an RNA fragment of a miR gene product that possesses one or more biological activities of a corresponding wild-type miR gene product. As described above, examples of such biological activities include, but are not limited to, inhibition of expression of a target RNA molecule and inhibition of a cellular process associated with solid cancer. In certain embodiments, the biologically-active fragment is at least about 5, 7, 10, 12, 15, or 17 nucleotides in length. In a particular embodiment, an isolated miR gene product can be administered to a subject in combination with one or more additional anti-cancer treatments. Suitable anti-cancer treatments include, but are not limited to, chemotherapy, radiation therapy and combinations thereof (e.g., chemoradiation).
When the at least one isolated miR gene product is up-regulated in the cancer cells, the method comprises administering to the subject an effective amount of at least one compound for inhibiting expression of the at least one miR gene product, referred to herein as miR gene expression-inhibition compounds, such that proliferation of solid cancer cells is inhibited. In a particular embodiment, the at least one miR expression-inhibition compound is specific for a miR gene product selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof. A miR gene expression-inhibiting compound can be administered to a subject in combination with one or more additional anti-cancer treatments. Suitable anti-cancer treatments include, but are not limited to, chemotherapy, radiation therapy and combinations thereof (e.g., chemoradiation).
The terms “treat”, “treating” and “treatment”, as used herein, refer to ameliorating symptoms associated with a disease or condition, for example, a solid cancer, including preventing or delaying the onset of the disease symptoms, and/or lessening the severity or frequency of symptoms of the disease or condition. The terms “subject”, “patient” and “individual” are defined herein to include animals, such as mammals, including, but not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species. In a preferred embodiment, the animal is a human.
As used herein, an “effective amount” of an isolated miR gene product is an amount sufficient to inhibit proliferation of a cancer cell in a subject suffering from a solid cancer. One skilled in the art can readily determine an effective amount of a miR gene product to be administered to a given subject, by taking into account factors, such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.
For example, an effective amount of an isolated miR gene product can be based on the approximate weight of a tumor mass to be treated. The approximate weight of a tumor mass can be determined by calculating the approximate volume of the mass, wherein one cubic centimeter of volume is roughly equivalent to one gram. An effective amount of the isolated miR gene product based on the weight of a tumor mass can be in the range of about 10-500 micrograms/gram of tumor mass. In certain embodiments, the tumor mass can be at least about 10 micrograms/gram of tumor mass, at least about 60 micrograms/gram of tumor mass or at least about 100 micrograms/gram of tumor mass.
An effective amount of an isolated miR gene product can also be based on the approximate or estimated body weight of a subject to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described herein. For example, an effective amount of the isolated miR gene product is administered to a subject can range from about 5 3000 micrograms/kg of body weight, from about 700-1000 micrograms/kg of body weight, or greater than about 1000 micrograms/kg of body weight.
One skilled in the art can also readily determine an appropriate dosage regimen for the administration of an isolated miR gene product to a given subject. For example, a miR gene product can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, a miR gene product can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more particularly from about seven to about ten days. In a particular dosage regimen, a miR gene product is administered once a day for seven days. Where a dosage regimen comprises multiple administrations, it is understood that the effective amount of the miR gene product administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.
As used herein, an “isolated” miR gene product is one that is synthesized, or altered or removed from the natural state through human intervention. For example, a synthetic miR gene product, or a miR gene product partially or completely separated from the coexisting materials of its natural state, is considered to be “isolated.” An isolated miR gene product can exist in substantially-purified form, or can exist in a cell into which the miR gene product has been delivered. Thus, a miR gene product that is deliberately delivered to, or expressed in, a cell is considered an “isolated” miR gene product. A miR gene product produced inside a cell from a miR precursor molecule is also considered to be an “isolated” molecule. According to the invention, the isolated miR gene products described herein can be used for the manufacture of a medicament for treating a solid cancer in a subject (e.g., a human).
Isolated miR gene products can be obtained using a number of standard techniques. For example, the miR gene products can be chemically synthesized or recombinantly produced using methods known in the art. In one embodiment, miR gene products are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical (part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research (Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem (Glasgow, UK).
Alternatively, the miR gene products can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 or H1 RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the miR gene products in cancer cells.
The miR gene products that are expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques. The miR gene products that are expressed from recombinant plasmids can also be delivered to, and expressed directly in, the cancer cells. The use of recombinant plasmids to deliver the miR gene products to cancer cells is discussed in more detail below.
The miR gene products can be expressed from a separate recombinant plasmid, or they can be expressed from the same recombinant plasmid. In one embodiment, the miR gene products are expressed as RNA precursor molecules from a single plasmid, and the precursor molecules are processed into the functional miR gene product by a suitable processing system, including, but not limited to, processing systems extant within a cancer cell. Other suitable processing systems include, e.g., the in vitro Drosophila cell lysate system (e.g., as described in U.S. Published Patent Application No. 2002/0086356 to Tuschl et al., the entire disclosure of which is incorporated herein by reference) and the E. coli RNAse III system (e.g., as described in U.S. Published Patent Application No. 2004/0014113 to Yang et al., the entire disclosure of which is incorporated herein by reference).
Selection of plasmids suitable for expressing the miR gene products, methods for inserting nucleic acid sequences into the plasmid to express the gene products, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are incorporated herein by reference.
In one embodiment, a plasmid expressing the miR gene products comprises a sequence encoding a miR precursor RNA under the control of the CMV intermediate-early promoter. As used herein, “under the control” of a promoter means that the nucleic acid sequences encoding the miR gene product are located 3′ of the promoter, so that the promoter can initiate transcription of the miR gene product coding sequences.
The miR gene products can also be expressed from recombinant viral vectors. It is contemplated that the miR gene products can be expressed from two separate recombinant viral vectors, or from the same viral vector. The RNA expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in cancer cells. The use of recombinant viral vectors to deliver the miR gene products to cancer cells is discussed in more detail below.
The recombinant viral vectors of the invention comprise sequences encoding the miR gene products and any suitable promoter for expressing the RNA sequences. Suitable promoters include, but are not limited to, the U6 or H1 RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the miR gene products in a cancer cell.
Any viral vector capable of accepting the coding sequences for the miR gene products can be used; for example, vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors that express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz, J. E., et al. (2002), J. Virol. 76:791-801, the entire disclosure of which is incorporated herein by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing RNA into the vector, methods of delivering the viral vector to the cells of interest, and recovery of the expressed RNA products are within the skill in the art. See, for example, Dornburg (1995), Gene Therapy 2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum. Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30, the entire disclosures of which are incorporated herein by reference.
Particularly suitable viral vectors are those derived from AV and AAV. A suitable AV vector for expressing the miR gene products, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia et al. (2002), Nat. Biotech. 20:1006-1010, the entire disclosure of which is incorporated herein by reference. Suitable AAV vectors for expressing the miR gene products, methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J. Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are incorporated herein by reference. In one embodiment, the miR gene products are expressed from a single recombinant AAV vector comprising the CMV intermediate early promoter.
In a certain embodiment, a recombinant AAV viral vector of the invention comprises a nucleic acid sequence encoding a miR precursor RNA in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter. As used herein, “in operable connection with a polyT termination sequence” means that the nucleic acid sequences encoding the sense or antisense strands are immediately adjacent to the polyT termination signal in the 5′ direction. During transcription of the miR sequences from the vector, the polyT termination signals act to terminate transcription.
In other embodiments of the treatment methods of the invention, an effective amount of at least one compound that inhibits miR expression can be administered to the subject. As used herein, “inhibiting miR expression” means that the production of the precursor and/or active, mature form of miR gene product after treatment is less than the amount produced prior to treatment. One skilled in the art can readily determine whether miR expression has been inhibited in a cancer cell, using, for example, the techniques for determining miR transcript level discussed above for the diagnostic method. Inhibition can occur at the level of gene expression (i.e., by inhibiting transcription of a miR gene encoding the miR gene product) or at the level of processing (e.g., by inhibiting processing of a miR precursor into a mature, active miR).
As used herein, an “effective amount” of a compound that inhibits miR expression is an amount sufficient to inhibit proliferation of a cancer cell in a subject suffering from a cancer (e.g., a solid cancer). One skilled in the art can readily determine an effective amount of a miR expression-inhibition compound to be administered to a given subject, by taking into account factors, such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.
For example, an effective amount of the expression-inhibition compound can be based on the approximate weight of a tumor mass to be treated, as described herein. An effective amount of a compound that inhibits miR expression can also be based on the approximate or estimated body weight of a subject to be treated, as described herein.
One skilled in the art can also readily determine an appropriate dosage regimen for administering a compound that inhibits miR expression to a given subject.
Suitable compounds for inhibiting miR gene expression include double-stranded RNA (such as short- or small-interfering RNA or “siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such as ribozymes. Each of these compounds can be targeted to a given miR gene product and interfere with the expression of (e.g., inhibit translation of, induce cleavage or destruction of) the target miR gene product. For example, expression of a given miR gene can be inhibited by inducing RNA interference of the miR gene with an isolated double-stranded RNA (“dsRNA”) molecule which has at least 90%, for example at least 95%, at least 98%, at least 99%, or 100%, sequence homology with at least a portion of the miR gene product. In a particular embodiment, the dsRNA molecule is a “short or small interfering RNA” or “siRNA.”
siRNA useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter “base-paired”). The sense strand comprises a nucleic acid sequence that is substantially identical to a nucleic acid sequence contained within the target miR gene product.
As used herein, a nucleic acid sequence in an siRNA which is “substantially identical” to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence, or that differs from the target sequence by one or two nucleotides. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded “hairpin” area.
The siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides.
One or both strands of the siRNA can also comprise a 3′ overhang. As used herein, a “3′ overhang” refers to at least one unpaired nucleotide extending from the 3′-end of a duplexed RNA strand. Thus, in certain embodiments, the siRNA comprises at least one 3′ overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, from 1 to about 5 nucleotides in length, from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length. In a particular embodiment, the 3′ overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the siRNA can comprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).
The siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for the isolated miR gene products. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. Published Patent Application No. 2002/0173478 to Gewirtz and in U.S. Published Patent Application No. 2004/0018176 to Reich et al., the entire disclosures of both of which are incorporated herein by reference.
Expression of a given miR gene can also be inhibited by an antisense nucleic acid. As used herein, an “antisense nucleic acid” refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA, RNA-DNA or RNA-peptide nucleic acid interactions, which alters the activity of the target RNA. Antisense nucleic acids suitable for use in the present methods are single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a miR gene product. The antisense nucleic acid can comprise a nucleic acid sequence that is 50-100% complementary, 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in a miR gene product. Nucleic acid sequences for the miR gene products are provided in Tables 1a and 1b. Without wishing to be bound by any theory, it is believed that the antisense nucleic acids activate RNase H or another cellular nuclease that digests the miR gene product/antisense nucleic acid duplex.
Antisense nucleic acids can also contain modifications to the nucleic acid backbone or to the sugar and base moieties (or their equivalent) to enhance target specificity, nuclease resistance, delivery or other properties related to efficacy of the molecule. Such modifications include cholesterol moieties, duplex intercalators, such as acridine, or one or more nuclease-resistant groups.
Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for the isolated miR gene products. Exemplary methods for producing and testing are within the skill in the art; see, e.g., Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 to Woolf et al., the entire disclosures of which are incorporated herein by reference.
Expression of a given miR gene can also be inhibited by an enzymatic nucleic acid. As used herein, an “enzymatic nucleic acid” refers to a nucleic acid comprising a substrate binding region that has complementarity to a contiguous nucleic acid sequence of a miR gene product, and which is able to specifically cleave the miR gene product. The enzymatic nucleic acid substrate binding region can be, for example, 50-100% complementary, 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in a miR gene product. The enzymatic nucleic acids can also comprise modifications at the base, sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.
The enzymatic nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for the isolated miR gene products. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in Werner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al. (1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No. 4,987,071 to Cech et al, the entire disclosures of which are incorporated herein by reference.
Administration of at least one miR gene product, or at least one compound for inhibiting miR expression, will inhibit the proliferation of cancer cells in a subject who has a solid cancer. As used herein, to “inhibit the proliferation of a cancer cell” means to kill the cell, or permanently or temporarily arrest or slow the growth of the cell. Inhibition of cancer cell proliferation can be inferred if the number of such cells in the subject remains constant or decreases after administration of the miR gene products or miR gene expression-inhibition compounds. An inhibition of cancer cell proliferation can also be inferred if the absolute number of such cells increases, but the rate of tumor growth decreases.
The number of cancer cells in the body of a subject can be determined by direct measurement, or by estimation from the size of primary or metastatic tumor masses. For example, the number of cancer cells in a subject can be measured by immunohistological methods, flow cytometry, or other techniques designed to detect characteristic surface markers of cancer cells.
The size of a tumor mass can be ascertained by direct visual observation, or by diagnostic imaging methods, such as X-ray, magnetic resonance imaging, ultrasound, and scintigraphy. Diagnostic imaging methods used to ascertain size of the tumor mass can be employed with or without contrast agents, as is known in the art. The size of a tumor mass can also be ascertained by physical means, such as palpation of the tissue mass or measurement of the tissue mass with a measuring instrument, such as a caliper.
The miR gene products or miR gene expression-inhibition compounds can be administered to a subject by any means suitable for delivering these compounds to cancer cells of the subject. For example, the miR gene products or miR expression-inhibition compounds can be administered by methods suitable to transfect cells of the subject with these compounds, or with nucleic acids comprising sequences encoding these compounds. In one embodiment, the cells are transfected with a plasmid or viral vector comprising sequences encoding at least one miR gene product or miR gene expression-inhibition compound.
Transfection methods for eukaryotic cells are well known in the art, and include, e.g., direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor-mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.
For example, cells can be transfected with a liposomal transfer compound, e.g., DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate, Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount of nucleic acid used is not critical to the practice of the invention; acceptable results may be achieved with 0.1-100 micrograms of nucleic acid/105 cells. For example, a ratio of about 0.5 micrograms of plasmid vector in 3 micrograms of DOTAP per 105 cells can be used.
A miR gene product or miR gene expression-inhibition compound can also be administered to a subject by any suitable enteral or parenteral administration route. Suitable enteral administration routes for the present methods include, e.g., oral, rectal, or intranasal delivery. Suitable parenteral administration routes include, e.g., intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. Particularly suitable administration routes are injection, infusion and direct injection into the tumor.
In the present methods, a miR gene product or miR gene product expression-inhibition compound can be administered to the subject either as naked RNA, in combination with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences that express the miR gene product or miR gene product expression-inhibition compound. Suitable delivery reagents include, e.g., the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), and liposomes.
Recombinant plasmids and viral vectors comprising sequences that express the miR gene products or miR gene expression-inhibition compounds, and techniques for delivering such plasmids and vectors to cancer cells, are discussed herein and/or are well known in the art.
In a particular embodiment, liposomes are used to deliver a miR gene product or miR gene expression-inhibition compound (or nucleic acids comprising sequences encoding them) to a subject. Liposomes can also increase the blood half-life of the gene products or nucleic acids. Suitable liposomes for use in the invention can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors, such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are incorporated herein by reference.
The liposomes for use in the present methods can comprise a ligand molecule that targets the liposome to cancer cells. Ligands that bind to receptors prevalent in cancer cells, such as monoclonal antibodies that bind to tumor cell antigens, are preferred.
The liposomes for use in the present methods can also be modified so as to avoid clearance by the mononuclear macrophage system (“MMS”) and reticuloendothelial system (“RES”). Such modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure. In a particularly preferred embodiment, a liposome of the invention can comprise both an opsonization-inhibition moiety and a ligand.
Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization-inhibiting moiety is “bound” to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly decreases the uptake of the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is incorporated herein by reference.
Opsonization-inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a number-average molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers, such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization-inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization-inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or a derivative thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called “PEGylated liposomes.”
The opsonization-inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture, such as tetrahydrofuran and water in a 30:12 ratio at 60° C.
Liposomes modified with opsonization-inhibition moieties remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called “stealth” liposomes. Stealth liposomes are known to accumulate in tissues fed by porous or “leaky” microvasculature. Thus, tissue characterized by such microvasculature defects, for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A., 18:6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation of the liposomes in the liver and spleen. Thus, liposomes that are modified with opsonization-inhibition moieties are particularly suited to deliver the miR gene products or miR gene expression-inhibition compounds (or nucleic acids comprising sequences encoding them) to tumor cells.
The miR gene products or miR gene expression-inhibition compounds can be formulated as pharmaceutical compositions, sometimes called “medicaments,” prior to administering them to a subject, according to techniques known in the art. Accordingly, the invention encompasses pharmaceutical compositions for treating a solid cancer. In one embodiment, the pharmaceutical composition comprises at least one isolated miR gene product, or an isolated variant or biologically-active fragment thereof, and a pharmaceutically-acceptable carrier. In a particular embodiment, the at least one miR gene product corresponds to a miR gene product that has a decreased level of expression in solid cancer cells relative to suitable control cells. In certain embodiments the isolated miR gene product is selected from the group consisting of miR-145, miR-155, miR-218-2 combinations thereof.
In other embodiments, the pharmaceutical compositions of the invention comprise at least one miR expression-inhibition compound. In a particular embodiment, the at least one miR gene expression-inhibition compound is specific for a miR gene whose expression is greater in solid cancer cells than control cells. In certain embodiments, the miR gene expression-inhibition compound is specific for one or more miR gene products selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, “pharmaceutical compositions” include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is incorporated herein by reference.
The present pharmaceutical compositions comprise at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising sequences encoding them) (e.g., 0.1 to 90% by weight), or a physiologically-acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. In certain embodiments, the pharmaceutical compositions of the invention additionally comprise one or more anti-cancer agents (e.g., chemotherapeutic agents). The pharmaceutical formulations of the invention can also comprise at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising sequences encoding them), which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. In one embodiment, the pharmaceutical composition comprises a miR gene or gene product that is not miR-15 and/or miR-16.
Especially suitable pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
In a particular embodiment, the pharmaceutical compositions of the invention comprise at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising sequences encoding them) that is resistant to degradation by nucleases. One skilled in the art can readily synthesize nucleic acids that are nuclease resistant, for example, by incorporating one or more ribonucleotides that is modified at the 2′-position into the miR gene product. Suitable 2′-modified ribonucleotides include those modified at the 2′-position with fluoro, amino, alkyl, alkoxy, and O-allyl.
Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
For solid pharmaceutical compositions of the invention, conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
For example, a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, preferably 25%-75%, of the at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising sequences encoding them). A pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, preferably 1%-10% by weight, of the at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising sequences encoding them) encapsulated in a liposome as described above, and a propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.
The pharmaceutical compositions of the invention can further comprise one or more anti-cancer agents. In a particular embodiment, the compositions comprise at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising sequences encoding them) and at least one chemotherapeutic agent. Chemotherapeutic agents that are suitable for the methods of the invention include, but are not limited to, DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial and exotoxic agents. Examples of suitable agents for the compositions of the present invention include, but are not limited to, cytidine arabinoside, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab, daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine, docetaxel, FOLFOX4.
The invention also encompasses methods of identifying an inhibitor of tumorigenesis, comprising providing a test agent to a cell and measuring the level of at least one miR gene product in the cell. In one embodiment, the method comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with decreased expression levels in cancer cells. An increase in the level of the miR gene product in the cell after the agent is provided, relative to a suitable control cell (e.g., agent is not provided), is indicative of the test agent being an inhibitor of tumorigenesis. In a particular embodiment, at least one miR gene product associated with decreased expression levels in cancer cells is selected from the group consisting of miR-145, miR-155, miR-218-2 and combinations thereof.
In other embodiments the method comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with increased expression levels in cancer cells. A decrease in the level of the miR gene product in the cell after the agent is provided, relative to a suitable control cell (e.g., agent is not provided), is indicative of the test agent being an inhibitor of tumorigenesis. In a particular embodiment, at least one miR gene product associated with increased expression levels in cancer cells is selected from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a.
Suitable agents include, but are not limited to drugs (e.g., small molecules, peptides), and biological macromolecules (e.g., proteins, nucleic acids). The agent can be produced recombinantly, synthetically, or it may be isolated (i.e., purified) from a natural source. Various methods for providing such agents to a cell (e.g., transfection) are well known in the art, and several of such methods are described hereinabove. Methods for detecting the expression of at least one miR gene product (e.g., Northern blotting, in situ hybridization, RT-PCR, expression profiling) are also well known in the art. Several of these methods are also described hereinabove.
The invention will now be illustrated by the following non-limiting examples.
EXEMPLIFICATION
The following Materials and Methods were used in the Examples:
Samples
A total of 540 samples, including 363 primary tumor samples and 177 normal tissues, were used in this study (Table 2). The following solid cancers were represented: lung carcinoma, breast carcinoma, prostate carcinoma, stomach carcinoma, colon carcinoma and pancreatic endocrine tumors. All samples were obtained with informed consent from each patient and were confirmed histologically. Normal samples were paired with samples from individuals affected with lung and stomach carcinoma, and from normal individuals for the remaining tissues. All normal breast samples were obtained by pooling 5 unrelated normal tissues. Total RNA was isolated from tissues using TRIzolTM reagent (Invitrogen), according to manufacturer's instructions.
MicroRNA Microarrays.
Microarray analysis was performed as previously described (Liu, C.-G., et al., Proc. Natl. Acad. Sci. USA 101: 11755-11760 (2004)). Briefly, 5 μg of total RNA was used for hybridization on miRNA microarray chips. These chips contain gene-specific 40-mer oligonucleotide probes, spotted by contacting technologies and covalently attached to a polymeric matrix. The microarrays were hybridized in 6×SSPE (0.9 M NaCl/60 mM NaH2PO4.H2O/8 mM EDTA, pH 7.4)/30% formamide at 25° C. for 18 hr, washed in 0.75×TNT (Tris.HCl/NaCl/Tween 20) at 37° C. for 40 min, and processed using direct detection of the biotin-labeled transcripts by streptavidin-Alexa647 (Molecular Probes) conjugate. Processed slides were scanned using a microarray scanner (GenePix Pro, Axon), with the laser set to 635 nm, at fixed PMT setting and a scan resolution of 10 mm. The data were confirmed by Northern blotting as described (Calin, G. A., et al., Proc. Natl. Acad. Sci. USA 101:11755-11760 (2004); Iorio, M. V., et al., Cancer Res. 65: 7065-7070 (2005)).







TABLE 2







Samples used in the study (tumors and corresponding normals).









Tumour type
Cancer Samples
Normal Samples












Lung carcinoma
123
123 


Breast carcinoma
79
 6*


Colon carcinoma
46
 8


Gastric carcinoma
20
21


Endocrine pancreatic tumours
39
12


Prostate cancer
56
 7


All tissues (527)
363
177 





*Pools of 5 unrelated normal breast tissues per sample (for a total of 30 unrelated individuals).






Computational Analysis.

Microarray images were analyzed using GenePix Pro (Axon). Average values of the replicate spots of each miRNA were background-subtracted, normalized and subjected to further analysis. Normalization was performed by using a per chip median normalization method, using the median array as a reference. Finally, miRNAs measured as present in at least the smallest of the two classes in a dataset were selected. Absent calls were thresholded to 4.5 prior to statistical analysis. This level is the average minimum intensity level detected in the experiments. MicroRNA nomenclature was according to the Genome Browser (www.genome.ucsc.edu) and the microRNA database at Sanger Center (Griffiths-Jones, S., Nucleic Acids Res 32: D109-11 (2004)); in case of discrepancies we followed the microRNA database. Differentially-expressed microRNAs were identified by using the t test procedure within significance analysis of microarrays (SAM)(Tusher, V. G., et al., Proc Natl Acad Sci USA 98: 5116-21 (2001). SAM calculates a score for each gene on the basis of the change in expression relative to the standard deviation of all measurements. Within SAM, t test was used. The microRNA signatures were determined by applying nearest shrunken centroids method. This method identifies a subgroup of genes that best characterizes each solid cancer from its respective normal counterpart. The prediction error was calculated by means of 10-fold cross validation, and for each cancer, we obtained the miR signature that resulted in the minimal prediction error. A resampling test was performed by random permutation analysis to compute the p-value of the shared signature.
EXAMPLE 1
Identification of a microRNA Expression Signature in Human Solid Cancers Statistics
The combined cancers/normal tissue comparison was conducted using a reduced number of lung samples (80 cancer and 40 normal samples), in order to balance the different tissues numerically, yielding a total of 404 samples. For statistical analysis, 137 miRs, whose expression values were above 256 (threshold value) in at least 50% of the samples, were retained from the 228 that were measured. A T test was used to identify differentially-expressed microRNAs (Table 3). The p-values of the T test were corrected for multiple testing procedures and to control Type I error rates. Adjusted p-values were obtained by performing resampling with 500,000 permutations (Jung, S. H., et al. Biostatistics 6: 157-69 (2005)). This analysis was performed in order to evaluate the results by using the same method as Lu and co-workers (Lu, J., et al., Nature 435: 834-8 (2005)).
As an alternative to T test, significance analysis of microarrays (SAM) was used to identify differentially-expressed microRNAs. This procedure allows for the control of false detection rate (FDR). The delta was chosen to result in an FDR less than or equal to 0.01. microRNA subsets which result in the best tumor classification, i.e., which best predict the two classes (cancer and normal), were then identified using the method of the nearest shrunken centroids, as implemented in PAM (prediction analysis of microarray). The prediction error was calculated by means of 10-fold cross validation. The microRNAs were selected yielding the minimum misclassification error after cross-validation.
Results
By T-test, 43 differentially-expressed miRs with an adjusted p-value below 0.05 were obtained (Table 3). Twenty six miRs were overexpressed and 17 were under-expressed relative to corresponding normal tissues when the six solid cancers are grouped together (breast, colon, lung, pancreas, prostate, stomach). These results indicated that the spectrum of expressed miRNAs in solid cancers is very different from that of normal cells (43 out of 137 miRNAs, 31%). Using SAM, 49 miRNAs were identified as differentially-expressed, of which 34 were up-regulated (Table 4). Using PAM, 36 over-expressed miRNAs in cancer (indicated by positive cancer scores) and 21 down-regulated miRs (indicated by negative cancer scores) were identified as differentially-expressed (Table 5). However, these analyses are not tailored to identify alterations in miR expression that consistently result in transformation, because miR expression is heavily tissue-specific (He, L., et al. Nature 435: 828-833 (2005); also see FIG. 1 and FIG. 2).
The clustering of miRs based on expression profiles derived from 363 solid cancer and 177 normal samples using 228 miRs is shown in FIG. 1. The tree, which shows a very good separation between the different tissues, was constructed using 137 different miRNAs that were expressed in at least 50% of the samples used in the study.







TABLE 3







Differentially regulated miRs in 6 solid cancer


types vs. normal tissues (T test stats.) *.















Cancer
Normal
Test




miR
ID
Mean
Mean
stat
Raw p
Adj p
















miR-21
#47
11.538663
9.648338
7.861136
2.00E−06
2.00E−06


miR-141
#137
9.024091
7.905398
6.238014
2.00E−06
2.00E−06


miR-212
#208
13.540651
14.33617
−6.57942
2.00E−06
2.00E−06


miR-128a prec
#113
12.32588
13.522675
−6.76388
2.00E−06
2.00E−06


miR-138-2
#133
11.739557
13.144746
−7.01204
2.00E−06
2.00E−06


miR-218-2
#221
11.279787
12.539366
−7.40557
2.00E−06
2.00E−06


miR-23b
#51
14.169748
15.949736
−8.37744
2.00E−06
2.00E−06


miR-195
#184
10.343991
9.172985
5.763262
2.00E−06
1.00E−05


miR-212 prec
#209
12.686966
13.661763
−5.83132
4.00E−06
1.00E−05


miR-29b-2
#95
11.27556
9.940731
5.660854
2.00E−06
1.40E−05


miR-199a-1
#191
10.032008
8.920183
5.528849
2.00E−06
3.00E−05


miR-9-3
#28
11.461922
12.570412
−5.43006
2.00E−06
4.60E−05


miR-128a
#114
13.024235
13.856624
−5.35102
6.00E−06
7.20E−05


let-7a-1
#1
12.616569
13.455246
−5.35346
2.00E−06
7.20E−05


let-7b
#5
13.42636
14.068521
−5.17701
1.00E−05
0.000146


miR-16-2
#39
10.460707
9.305895
5.048375
4.00E−06
0.000224


miR-199a-2
#192
9.714225
8.759237
4.862553
1.00E−05
0.000494


miR-152 prec
#151
11.388676
12.357529
−4.83716
2.00E−06
0.00053


miR-16-1
#38
10.443169
9.338182
4.755258
1.00E−05
0.00071


miR-30d
#72
13.982017
14.775206
−4.5707
1.20E−05
0.001476


miR-34a
#78
10.675566
9.63769
4.467301
2.60E−05
0.00217


miR-17-5p
#41
11.567244
10.281468
4.341834
3.80E−05
0.0034


miR-128b
#115
10.930395
9.947746
4.304764
3.80E−05
0.003912


miR-20a
#46
11.409852
10.19284
4.304678
3.20E−05
0.003912


miR-181b-1 prec
#211
9.577504
8.804294
4.285968
4.80E−05
0.004126


miR-132
#121
9.599947
8.775966
4.284737
5.60E−05
0.004126


miR-200b
#195
9.475221
8.527243
4.221511
4.00E−05
0.0052


let-7a-3
#4
10.436089
9.511546
4.08952
0.000104
0.008242


miR-138-1
#132
8.299613
9.200253
−4.05204
5.60E−05
0.00931


miR-29c
#65
11.291005
10.326912
4.019385
0.000144
0.010312


miR-29a
#62
11.381359
10.461075
4.013697
0.00015
0.010398


miR-96
#86
11.37218
12.136636
−3.94825
0.000138
0.012962


miR-191
#177
13.498207
12.729872
3.817228
0.000158
0.02015


miR-27a
#59
10.399338
9.548582
3.715048
0.000344
0.028096


let-7g
#15
10.819688
10.01157
3.653239
0.000426
0.033874


miR-9-1
#24
10.102819
9.212988
3.651886
0.000388
0.033874


miR-125a
#107
10.960998
10.005312
3.651356
0.000452
0.033874


miR-95
#84
9.435733
8.751331
3.59406
0.000478
0.039594


miR-155
#157
12.505359
13.231221
−3.58369
0.000614
0.040394


miR-199b
#194
9.755066
9.082751
3.55934
0.000588
0.04314


miR-24-2
#54
12.611696
11.612557
3.518774
0.00087
0.048278


let-7e
#11
12.497795
13.055093
−3.51589
0.00054
0.048354


miR-92-1
#81
16.081074
16.592426
−3.50446
0.000928
0.049828





* Forty-three miRs have an adjusted p-value lower than 0.05. Twenty-six miRs are overexpressed and 17 down-regulated in breast, colon, lung, pancreas, prostate, stomach carcinomas.













TABLE 4







Differentially regulated miRs in 6 solid cancer types vs. normal


tissues (SAM, significance analysis of microarrays) *.















d.

p.
q.
R.


miR
ID
value
stdev
value
value
fold
















miR-21
#47
3.156
0.24
0
0
2.593


miR-23b
#51
−3.117
0.212
0
0
0.443


miR-138-2
#133
−2.514
0.2
0
0
0.402


miR-218-2
#221
−2.383
0.17
0
0
0.384


miR-29b-2
#95
2.246
0.236
0
0
1.868


miR-128a prec
#113
−2.235
0.177
0
0
0.368


miR-195
#184
2.085
0.203
0
0
1.695


miR-141
#137
2.08
0.179
0
0
2.459


miR-199a-1
#191
1.987
0.201
0
0
1.945


miR-9-3
#28
−1.97
0.204
0
0
0.433


miR-16-2
#39
1.966
0.229
0
0
1.788


miR-17-5p
#41
1.964
0.296
0
0
0.725


miR-20a
#46
1.898
0.283
0
0
0.969


miR-16-1
#38
1.87
0.232
0
0
1.447


miR-212 prec
#209
−1.854
0.167
0
0
0.509


miR-34a
#78
1.756
0.232
0
0
1.219


miR-152 prec
#151
−1.734
0.2
0
0
0.46


miR-199a-2
#192
1.721
0.196
0
0
1.838


miR-128b
#115
1.674
0.228
0
0
1.266


miR-212
#208
−1.659
0.121
0
0
0.627


let-7a-1
#1
−1.628
0.157
0
0
0.461


miR-200b
#195
1.626
0.225
0
0
1.432


miR-128a
#114
−1.619
0.156
0
0
0.511


miR-29c
#65
1.611
0.24
0
0
1.225


let-7a-3
#4
1.581
0.226
0
0
1.109


miR-29a
#62
1.565
0.229
0
0
1.706


miR-24-2
#54
1.555
0.284
0
0
0.831


miR-138-1
#132
−1.551
0.222
0
0
0.432


miR-125a
#107
1.541
0.262
0
0
1.164


miR-106a
#99
1.514
0.275
0
0
0.952


miR-132
#121
1.496
0.192
0
0
2.158


miR-30d
#72
−1.491
0.174
0
0
0.424


miR-9-1
#24
1.478
0.244
0
0
0.763


miR-27a
#59
1.448
0.229
0
0
1.174


miR-181b-1 prec
#211
1.435
0.18
0
0
1.525


let-7g
#15
1.394
0.221
0
0
1.072


miR-96
#86
−1.384
0.194
0
0
0.519


miR-191
#177
1.372
0.201
0
0
1.165


miR-93-1
#83
1.363
0.266
0
0
0.775


miR-136
#130
−1.355
0.267
0
0
0.364


miR-205
#201
1.343
0.309
0
0
1.281


miR-185
#170
1.287
0.222
0.001
0.001
0.609


miR-125b-1
#109
1.262
0.283
0.001
0.001
1.215


miR-10a
#30
1.252
0.227
0.001
0.001
1.643


miR-95
#84
1.247
0.19
0.001
0.001
1.509


miR-199b
#194
1.228
0.189
0.001
0.001
1.246


miR-10b
#32
1.219
0.232
0.002
0.001
1.342


let-7i
#10
1.216
0.203
0.002
0.001
1.026


miR-210
#205
1.213
0.237
0.002
0.001
1.088





* Thirty five miRs are over-expressed and 14 are down-regulated in breast, colon, lung, pancreas, prostate, stomach carcinomas (Delta = 0.9, FDR = 0.001).













TABLE 5







MicroRNAs selected by PAM (prediction analysis of microarray)


in 6 solid cancer types vs. normal tissues










miR
ID
Solid cancer score
Normal tissues score













miR-21
#47
0.0801
−0.2643


miR-138-2
#133
−0.055
0.1815


miR-218-2
#221
−0.0535
0.1765


miR-23b
#51
−0.0516
0.17


miR-128a prec
#113
−0.0498
0.1642


miR-29b-2
#95
0.0457
−0.1508


miR-195
#184
0.0404
−0.1333


miR-17-5p
#41
0.0383
−0.1263


miR-9-3
#28
−0.0357
0.1176


miR-212 prec
#209
−0.0342
0.1129


miR-20a
#46
0.0322
−0.1061


miR-141
#137
0.0322
−0.1061


miR-199a-1
#191
0.0319
−0.1053


miR-16-2
#39
0.0315
−0.1037


miR-152 prec
#151
−0.0283
0.0933


miR-16-1
#38
0.0277
−0.0913


miR-34a
#78
0.0269
−0.0886


miR-212
#208
−0.0265
0.0875


let-7a-1
#1
−0.0264
0.0872


miR-128a
#114
−0.0259
0.0855


miR-128b
#115
0.0254
−0.0839


miR-24-2
#54
0.0244
−0.0803


miR-29c
#65
0.0224
−0.0738


miR-199a-2
#192
0.0223
−0.0736


let-7a-3
#4
0.0221
−0.073


miR-191
#177
0.0188
−0.062


miR-125a
#107
0.0186
−0.0613


miR-30d
#72
−0.0185
0.061


miR-29a
#62
0.0184
−0.0608


miR-106a
#99
0.0177
−0.0584


miR-93-1
#83
0.0163
−0.0537


miR-200b
#195
0.0159
−0.0524


let-7g
#15
0.0158
−0.0521


miR-27a
#59
0.0157
−0.0518


miR-96
#86
−0.0156
0.0514


let-7b
#5
−0.0152
0.0501


miR-138-1
#132
−0.0151
0.0499


miR-9-1
#24
0.0136
−0.0448


miR-181b-1 prec
#211
0.0134
−0.0442


miR-155
#157
−0.0128
0.0423


miR-132
#121
0.0127
−0.0418


miR-136
#130
−0.0112
0.037


let-7i
#10
0.0103
−0.034


miR-210
#205
0.0074
−0.0245


miR-205
#201
0.0073
−0.024


*miR-185
#170
0.0071
−0.0234


miR-24-1
#52
0.007
−0.023


miR-199b
#194
0.0064
−0.021


miR-125b-1
#109
0.006
−0.0199


miR-206 prec
#203
−0.005
0.0166


miR-10a
#30
0.0045
−0.015


miR-95
#84
0.0045
−0.0149


let-7c
#11
−0.0039
0.013


miR-124a-3
#106
−0.0028
0.0091


miR-10b
#32
0.002
−0.0066


miR-185 prec
#171
−0.0014
0.0047


miR-92-1
#81
−2.00E−04
5.00E−04





*T = 1.5 and misclassification error = 0.176. Thirty six over-expressed miRs in cancer are indicated by positive cancer scores; 21 down-regulated miRs are indicated by negative cancer scores.






EXAMPLE 2
Identification of microRNA Expression Signatures Associated with Various Human Solid Cancers
Results
To identify microRNAs that are prognostic for cancer status associated with solid tumors, without incurring bias due to tissue specificity, an alternative approach was used. First, six tissue-specific signatures, one for each cancer histotype, were obtained by performing independent PAM tests (summarized in Tables 6 and 7) Specific signatures for each cancer are shown in Tables 8-13: e.g., breast—Table 8; colon—Table 9; lung—Table 10; pancreas—Table 11; prostate—Table 12; stomach—Table 13. Using these data, deregulated microRNAs that were shared among the different histotype miRNA signatures were identified (Table 14). In order to compute the p-values for this comparative analysis, a re-sampling test with 1,000,000 random permutations on the miRNA identity was performed. The p-value was defined as the relative frequency of simulation scores exceeding the real score. Twenty-one misregulated microRNAs that were common to at least 3 types of solid cancers (p-value=2.5×10−3) were identified (Table 14).







TABLE 6







MicroRNAs used to classify human cancers and normal tissues*.













Up-
Down-
Misclassification




regulated
regulated
error after 10 fold



Cancer
miRs
miRs
cross validation
















Breast
15
12
0.08



Colon
21
1
0.09



Lung
35
3
0.31



Pancreas
55
2
0.02



Prostate
39
6
0.11



Stomach
22
6
0.19







*Median normalization was performed and the method of the nearest shrunken centroids was used to select predictive miRNAs.













TABLE 7







Deregulated microRNAs in solid common cancers*.












PAM Up-
SAM Up-
PAM Down-
SAM Down-


Cancer
regulated
regulated
regulated
regulated














Breast
15
 3 (FDR = 0.33)
12
47


Colon
21
42 (FDR <= 0.06)
1
5


Lung
35
38 (FDR <= 0.01)
3
3


Pancreas
55
50 (FDR <= 0.01)
2
8


Stomach
22
22 (FDR = 0.06)
6
4


Prostate
39
49 (FDR = 0.06)
6
3





*Prediction analysis of microarrays (PAM) identifies those genes which best characterize cancers and normal tissues, whilst significance analysis of microarrays (SAM) identifies all those which have differential expression in the two classes. False detection rates (FDR) computed in SAM are indicated in parenthesis.













TABLE 8







MicroRNAs selected by prediction analysis of microarray


(PAM) in breast cancer (cancer vs. normal tissues) *.











miR
Cancer score
Normal score















miR-21 (#47)
0.0331
−0.4364



miR-29b-2 (#95)
0.0263
−0.3467



miR-146 (#144)
0.0182
−0.2391



miR-125b-2 (#111)
−0.0174
0.2286



miR-125b-1 (#109)
−0.0169
0.222



miR-10b (#32)
−0.0164
0.2166



miR-145 (#143)
−0.0158
0.2076



miR-181a (#158)
0.0153
−0.201



miR-140 (#136)
−0.0122
0.1613



miR-213 (#160)
0.0116
−0.1527



miR-29a prec (#63)
0.0109
−0.1441



miR-181b-1 (#210)
0.0098
−0.1284



miR-199b (#194)
0.0089
−0.1172



miR-29b-1 (#64)
0.0084
−0.1111



miR-130a (#120)
−0.0076
0.1001



miR-155 (#157)
0.0072
−0.0951



let-7a-2 (#3)
−0.0042
0.0554



miR-205 (#201)
−0.004
0.0533



miR-29c (#65)
0.0032
−0.0423



miR-224 (#228)
−0.003
0.0399



miR-100 (#91)
−0.0021
0.0283



miR-31 (#73)
0.0017
−0.022



miR-30c (#70)
−7.00E−04  
0.009



miR-17-5p (#41)
7.00E−04
−0.0089



miR-210 (#205)
4.00E−04
−0.0057



miR-122a (#101)
4.00E−04
−0.005



miR-16-2 (#39)
−1.00E−04  
0.0013







* 27 miRs selected, misclassification error after cross validation of 0.008. Seventeen overexpressed miRs in cancer are indicated by positive cancer scores; 12 down-regulated miRs are indicated by negative cancer scores.













TABLE 9







MicroRNAs selected by prediction analysis of microarray


(PAM) in colon (cancer vs. normal tissues)*.











miR
Cancer score
Normal score















miR-24-1 (#52)
0.0972
−0.5589



miR-29b-2 (#95)
0.0669
−0.3845



miR-20a (#46)
0.0596
−0.3424



miR-10a (#30)
0.0511
−0.2938



miR-32 (#75)
0.0401
−0.2306



miR-203 (#197)
0.0391
−0.2251



miR-106a (#99)
0.0364
−0.2094



miR-17-5p (#41)
0.0349
−0.2005



miR-30c (#70)
0.0328
−0.1888



miR-223 (#227)
0.0302
−0.1736



miR-126* (#102)
0.0199
−0.1144



miR-128b (#115)
0.0177
−0.102



miR-21 (#47)
0.0162
−0.0929



miR-24-2 (#54)
0.0145
−0.0835



miR-99b prec (#88)
0.0125
−0.0721



miR-155 (#157)
0.0092
−0.0528



miR-213 (#160)
0.0091
−0.0522



miR-150 (#148)
0.0042
−0.0243



miR-107 (#100)
0.003
−0.0173



miR-191 (#177)
0.0028
−0.0159



miR-221 (#224)
0.002
−0.0116



miR-9-3 (#28)
−0.0014
0.0083







*22 miRs selected, misclassification error after cross validation of 0.09. Twenty-one over-expressed miRs in cancer are indicated by positive cancer scores; 1 down-regulated miR is indicated by a negative cancer score.













TABLE 10







MicroRNAs selected by prediction analysis of microarray


(PAM) in lung cancer (cancer vs. normal tissues)*.











miR
Cancer score
Normal score















miR-21 (#47)
0.175
−0.175



miR-205 (#201)
0.1317
−0.1317



miR-200b (#195)
0.1127
−0.1127



miR-9-1 (#24)
0.1014
−0.1014



miR-210 (#205)
0.0994
−0.0994



miR-148 (#146)
0.0737
−0.0737



miR-141 (#137)
0.0631
−0.0631



miR-132 (#121)
0.0586
−0.0586



miR-215 (#213)
0.0575
−0.0575



miR-128b (#115)
0.0559
−0.0559



let-7g (#15)
0.0557
−0.0557



miR-16-2 (#39)
0.0547
−0.0547



miR-129-1/2 prec (#118)
0.0515
−0.0515



miR-126* (#102)
−0.0406
0.0406



miR-142-as (#139)
0.0366
−0.0366



miR-30d (#72)
−0.0313
0.0313



miR-30a-5p (#66)
−0.0297
0.0297



miR-7-2 (#21)
0.0273
−0.0273



miR-199a-1 (#191)
0.0256
−0.0256



miR-127 (#112)
0.0254
−0.0254



miR-34a prec (#79)
0.0214
−0.0214



miR-34a (#78)
0.0188
−0.0188



miR-136 (#130)
0.0174
−0.0174



miR-202 (#196)
0.0165
−0.0165



miR-196-2 (#188)
0.0134
−0.0134



miR-199a-2 (#192)
0.0126
−0.0126



let-7a-2 (#3)
0.0109
−0.0109



miR-124a-1 (#104)
0.0081
−0.0081



miR-149 (#147)
0.0079
−0.0079



miR-17-5p (#41)
0.0061
−0.0061



miR-196-1 prec (#186)
0.0053
−0.0053



miR-10a (#30)
0.0049
−0.0049



miR-99b prec (#88)
0.0045
−0.0045



miR-196-1 (#185)
0.0044
−0.0044



miR-199b (#194)
0.0039
−0.0039



miR-191 (#177)
0.0032
−0.0032



miR-195 (#184)
7.00E−04
−7.00E−04



miR-155 (#157)
7.00E−04
−7.00E−04







*38 miRs selected, misclassification error after cross validation of 0.31. Thirty-five over-expressed miRs in cancer are indicated by positive cancer scores; 3 down-regulated miRs are indicated by negative cancer scores.













TABLE 11







MicroRNAs selected by prediction analysis of microarray


(PAM) in pancreatic cancer (cancer vs. normal tissues)*.











miR
Cancer score
Normal score















miR-103-2 (#96)
0.4746
−1.582



miR-103-1 (#97)
0.4089
−1.3631



miR-24-2 (#54)
0.4059
−1.3529



miR-107 (#100)
0.3701
−1.2336



miR-100 (#91)
0.3546
−1.182



miR-125b-2 (#111)
0.3147
−1.0489



miR-125b-1 (#109)
0.3071
−1.0237



miR-24-1 (#52)
0.2846
−0.9488



miR-191 (#177)
0.2661
−0.887



miR-23a (#50)
0.2586
−0.8619



miR-26a-1 (#56)
0.2081
−0.6937



miR-125a (#107)
0.1932
−0.644



miR-130a (#120)
0.1891
−0.6303



miR-26b (#58)
0.1861
−0.6203



miR-145 (#143)
0.1847
−0.6158



miR-221 (#224)
0.177
−0.59



miR-126* (#102)
0.1732
−0.5772



miR-16-2 (#39)
0.1698
−0.5659



miR-146 (#144)
0.1656
−0.552



miR-214 (#212)
0.1642
−0.5472



miR-99b (#89)
0.1636
−0.5454



miR-128b (#115)
0.1536
−0.512



miR-155 (#157)
−0.1529
0.5098



miR-29b-2 (#95)
0.1487
−0.4956



miR-29a (#62)
0.1454
−0.4848



miR-25 (#55)
0.1432
−0.4775



miR-16-1 (#38)
0.1424
−0.4746



miR-99a (#90)
0.1374
−0.4581



miR-224 (#228)
0.1365
−0.4549



miR-30d (#72)
0.1301
−0.4336



miR-92-2 (#82)
0.116
−0.3865



miR-199a-1 (#191)
0.1158
−0.3861



miR-223 (#227)
0.1141
−0.3803



miR-29c (#65)
0.113
−0.3768



miR-30b (#68)
0.1008
−0.3361



miR-129-1/2 (#117)
0.1001
−0.3337



miR-197 (#189)
0.0975
−0.325



miR-17-5p (#41)
0.0955
−0.3185



miR-30c (#70)
0.0948
−0.316



miR-7-1 (#19)
0.0933
−0.311



miR-93-1 (#83)
0.0918
−0.3061



miR-140 (#136)
0.0904
−0.3015



miR-30a-5p (#66)
0.077
−0.2568



miR-132 (#121)
0.0654
−0.2179



miR-181b-1 (#210)
0.0576
−0.1918



miR-152 prec (#151)
−0.0477
0.1591



miR-23b (#51)
0.0469
−0.1562



miR-20a (#46)
0.0452
−0.1507



miR-222 (#225)
0.0416
−0.1385



miR-27a (#59)
0.0405
−0.1351



miR-92-1 (#81)
0.0332
−0.1106



miR-21 (#47)
0.0288
−0.0959



miR-129-1/2 prec (#118)
0.0282
−0.0939



miR-150 (#148)
0.0173
−0.0578



miR-32 (#75)
0.0167
−0.0558



miR-106a (#99)
0.0142
−0.0473



miR-29b-1 (#64)
0.0084
−0.028







*57 miRs selected, misclassification error after cross validation of 0.02. Fifty-seven miRs are over-expressed and 2 are down-regulated in cancer (indicated by positive and negative scores, respectively).













TABLE 12







MicroRNAs selected by prediction analysis of microarray


(PAM) in prostate cancer (cancer vs. normal tissues) *.











miR
Cancer score
Normal score















let-7d (#8)
0.0528
−0.4227



miR-128a prec (#113)
−0.0412
0.3298



miR-195 (#184)
0.04
−0.3199



miR-203 (#197)
0.0356
−0.2851



let-7a-2 prec (#2)
−0.0313
0.2504



miR-34a (#78)
0.0303
−0.2428



miR-20a (#46)
0.029
−0.2319



miR-218-2 (#221)
−0.0252
0.2018



miR-29a (#62)
0.0247
−0.1978



miR-25 (#55)
0.0233
−0.1861



miR-95 (#84)
0.0233
−0.1861



miR-197 (#189)
0.0198
−0.1587



miR-135-2 (#128)
0.0198
−0.1582



miR-187 (#173)
0.0192
−0.1535



miR-196-1 (#185)
0.0176
−0.1411



miR-148 (#146)
0.0175
−0.1401



miR-191 (#177)
0.017
−0.136



miR-21 (#47)
0.0169
−0.1351



let-7i (#10)
0.0163
−0.1303



miR-198 (#190)
0.0145
−0.1161



miR-199a-2 (#192)
0.0136
−0.1088



miR-30c (#70)
0.0133
−0.1062



miR-17-5p (#41)
0.0132
−0.1053



miR-92-2 (#82)
0.012
−0.0961



miR-146 (#144)
0.0113
−0.0908



miR-181b-1 prec (#211)
0.011
−0.0878



miR-32 (#75)
0.0109
−0.0873



miR-206 (#202)
0.0104
−0.083



miR-184 prec (#169)
0.0096
−0.0764



miR-29a prec (#63)
−0.0095
0.076



miR-29b-2 (#95)
0.0092
−0.0739



miR-149 (#147)
−0.0084
0.0676



miR-181b-1 (#210)
0.0049
−0.0392



miR-196-1 prec (#186)
0.0042
−0.0335



miR-93-1 (#83)
0.0039
−0.0312



miR-223 (#227)
0.0038
−0.0308



miR-16-1 (#38)
0.0028
−0.0226



miR-101-1 prec (#92)
0.0015
−0.0123



miR-124a-1 (#104)
0.0015
−0.0119



miR-26a-1 (#56)
0.0015
−0.0119



miR-214 (#212)
0.0013
−0.0105



miR-27a (#59)
0.0011
−0.0091



miR-24-1 (#53)
−8.00E−04  
0.0067



miR-106a (#99)
7.00E−04
−0.0057



miR-199a-1 (#191)
4.00E−04
−0.0029







* T = 1, 45 miRs selected, misclassification error after cross validation of 0.11. Thirty-nine over-expressed miRs in cancer are indicated by positive cancer scores; 6 downregulated miRs are indicated by negative cancer scores.













TABLE 13







MicroRNAs selected by prediction analysis of microarray


(PAM) in stomach cancer (cancer vs. normal tissues) *.











miR
Cancer score
Normal score















miR-223 (#227)
0.1896
−0.1806



miR-21 (#47)
0.1872
−0.1783



miR-218-2 (#221)
−0.1552
0.1478



miR-103-2 (#96)
0.1206
−0.1148



miR-92-2 (#82)
0.1142
−0.1088



miR-25 (#55)
0.1097
−0.1045



miR-136 (#130)
−0.1097
0.1045



miR-191 (#177)
0.0946
−0.0901



miR-221 (#224)
0.0919
−0.0876



miR-125b-2 (#111)
0.0913
−0.0869



miR-103-1 (#97)
0.0837
−0.0797



miR-214 (#212)
0.0749
−0.0713



miR-222 (#225)
0.0749
−0.0713



miR-212 prec (#209)
−0.054
0.0514



miR-125b-1 (#109)
0.0528
−0.0503



miR-100 (#91)
0.0526
−0.0501



miR-107 (#100)
0.0388
−0.0369



miR-92-1 (#81)
0.0369
−0.0351



miR-96 (#86)
−0.0306
0.0291



miR-192 (#178)
0.0236
−0.0224



miR-23a (#50)
0.022
−0.021



miR-215 (#213)
0.0204
−0.0194



miR-7-2 (#21)
0.0189
−0.018



miR-138-2 (#133)
−0.0185
0.0176



miR-24-1 (#52)
0.0151
−0.0144



miR-99b (#89)
0.0098
−0.0093



miR-33b (#76)
−0.0049
0.0046



miR-24-2 (#54)
0.0041
−0.0039







* T = 1, 28 miRs selected, misclassification error after cross validation of 0.19. Twenty-two over-expressed miRs in cancer are indicated by positive cancer scores; 6 down-regulated miRs are indicated by negative cancer scores.













TABLE 14







The microRNAs shared by the signatures of the 6 solid cancers*.









miR
N
Tumor Type





miR-21
6
Breast Colon Lung Pancreas Prostate Stomach


miR-17-5p
5
Breast Colon Lung Pancreas Prostate


miR-191
5
Colon Lung Pancreas Prostate Stomach


miR-29b-2
4
Breast Colon Pancreas Prostate


miR-223
4
Colon Pancreas Prostate Stomach


miR-128b
3
Colon Lung Pancreas


miR-199a-1
3
Lung Pancreas Prostate


miR-24-1
3
Colon Pancreas Stomach


miR-24-2
3
Colon Pancreas Stomach


miR-146
3
Breast Pancreas Prostate


miR-155
3
Breast Colon Lung


miR-181b-1
3
Breast Pancreas Prostate


miR-20a
3
Colon Pancreas Prostate


miR-107
3
Colon Pancreas Stomach


miR-32
3
Colon Pancreas Prostate


miR-92-2
3
Pancreas Prostate Stomach


miR-214
3
Pancreas Prostate Stomach


miR-30c
3
Colon Pancreas Prostate


miR-25
3
Pancreas Prostate Stomach


miR-221
3
Colon Pancreas Stomach


miR-106a
3
Colon Pancreas Prostate





*The list includes 21 commonly up-regulated microRNAs in 3 or more (N) types of solid cancers (p-value = 2.5 × 10−3).






To maximize concision, the mean absolute expression levels of the deregulated miRs for the 6 cancer/normal pairs were computed. Using the expression level of miRs in the comprehensive subset, the different tissues were correctly classified, irrespective of the disease status (FIG. 3).
FIG. 4 shows differential expression of the common microRNAs across the different tumor tissues, in relation to the normal tissues. The tree displays the different cancer types according to fold changes in the miRNA subset. Prostate, colon, stomach and pancreatic tissues are most similar among them, while lung and breast tissues were represented by a fairly different signature (FIG. 4). This tree clearly shows which miRNAs are associated with a particular cancer histotype.
Strikingly, miR-21, miR-191 and miR-17-5p are significantly over-expressed in all, or in 5 out of 6, of the tumor types that were considered. miR-21 was reported to be over-expressed in glioblastoma and to have anti-apoptotic properties (Chan, J. A., et al., Cancer Res. 65: 6029-6033 (2005)). Lung cancer shares a portion of its signature with breast cancer and a portion with the other solid tumors, including miR-17/20/92, all three of which are members of the microRNA cluster that actively cooperates with c-Myc to accelerate lymphomagenesis (He, L., et al., Nature 435: 828-833 (2005)). The identification of these microRNAs as being over-expressed is an excellent confirmation of our approach. A second miRNA group that is activated includes miR-210 and miR-213, together with miR-155, which was already reported to be amplified in large cell lymphomas (E is, P. S., et al., Proc. Natl. Acad. Sci. USA 102: 3627-3632 (2005)), children with Burkitt lymphoma (Metzler, M., et al., Genes Chromosomes Cancer 39:167-169 (2004)) and various B cell lymphomas (Kluiver, J, et al., J. Pathol., e-published online, Jul. 22, 2005). These microRNAs are the only ones up-regulated in breast and lung cancer. miR-218-2 is consistently down-regulated in colon, stomach, prostate and pancreas cancers, but not in lung and breast carcinomas.
Several observations strengthen these results. First, in this study, the expression levels of both the precursor pre-miRNA and the mature miRNA were determined for the majority of genes. Of note, with the exception of miR-212 and miR-128a, in all other instances, the abnormally-expressed region was that corresponding to the active gene product. Second, as shown in FIG. 3, the expression variation of the miRNAs in the comprehensive subset was often univocal (namely, down- or up-regulation) across the different types of cancers, suggesting a common mechanism in human tumorigenesis. Third, the microarray data were validated by solution hybridization for 12 breast samples (miR-125b, miR-145 and miR-21; Iorio, M. V., et al., Cancer Res. 65: 7065-7070 (2005)) and 17 endocrine pancreatic and normal samples (miR-103, miR-155 and miR-204; data not shown), strongly confirming the accuracy of the microarray data.
EXAMPLE 3
Identification of Predicted Targets for microRNAs that are Deregulated in Solid Tumors
Materials and Methods:
Tumor Suppressor and Oncogene Target Predictions
The most recent TargetScan predictions (April 2005) were used to identify putative microRNA targets. These include essentially the 3′UTR targets reported by Lewis et al. (Lewis, B. P., et al, Cell 120: 15-20 (2005)), with a few changes arising from updated gene boundary definitions from the April 2005 UCSC Genome Browser mapping of RefSeq mRNAs to the hg17 human genome assembly. Among the putative targets, known cancer genes (tumor suppressors and oncogenes) were specified according to their identification in the Cancer Gene Census, which is accessible at the internet site www.sanger.ac.uk/genetics/CGP/Census/, or as reported by OMIM at www.ncbi.nlm.nih.gov.
Target In Vitro Assays
For luciferase reporter experiments, 3′ UTR segments of Rb1, TGFBR2 and Plag1 that are predicted to interact with specific cancer-associated microRNAs were amplified by PCR from human genomic DNA and inserted into the pGL3 control vector (Promega) using the XbaI site immediately downstream from the stop codon of luciferase. The human megakaryocytic cell line, MEG-01, was grown in 10% FBS in RPMI medium 1640, supplemented with 1× nonessential amino acid and 1 mmol sodium pyruvate at 37° C. in a hunified atmosphere of 5% CO2. The cells were co-transfected in 12-well plates by using siPORT neoFX (Ambion, Austin, Tex.), according to the manufacturer's protocol, with 0.4 mg of the firefly luciferase reporter vector and 0.08 μg of the control vector containing Renilla luciferase, pRL-TK (Promega). For each well, microRNA oligonucleotides (Dharmacon Research, Lafayette, Colo.) and anti-sense or scrambled oligonucleotides (Ambion) were used at a concentration of 10 nM. Firefly and Renilla luciferase activities were measured consecutively at 24 h post transfection using dual-luciferase assays (Promega).
Western Blotting for RB1
Levels of RB1 protein were quantified using a mouse monoclonal anti-RB1 antibody (Santa Cruz, Calif.) using standard procedures for Western blotting. The normalization was performed with mouse monoclonal anti-Actin antibody (Sigma).
Results
The functional significance of microRNA deregulation in cancer needs to be understood. In solid tumors, it appears that the most common microRNA event is gain of expression, while loss of expression in cancer is a more limited event, and more tissue specific. We used a three-step consequential approach in the following order: first, “in silico” prediction of targets, then luciferase assay for first validation of cancer relevant targets and finally, ex vivo tumor correlation between miRNA expression (by microarray) and target protein expression (by Western blotting) for a specific miRNA:mRNA interactor pair. Relevant targets for cancer miRNAs could be either recessive (e.g., tumor suppressors) or dominant (e.g., oncogenes) cancer genes. To test the hypothesis that microRNAs that are deregulated in solid tumors target known oncogenes or tumor suppressors, the predicted targets for these miRNAs were determined using TargetScan, a database of conserved 3′ UTR microRNA targets (Lewis, B. P., et al, Cell 120: 15-20 (2005)). TargetScan contained 5,121 predictions for 18 miRNAs that are dysregulated in solid tumors, in the total 22,402 (26.5%) predictions. One hundred fifteen out of 263 (44%) well-known cancer genes were predicted as targets for these 18 miRNAs (Table 15). Because a high percentage of cancer genes are targeted by miRs that are deregulated in solid tumors, it is unlikely that these predictions are due to chance (P<0.0001 at Fisher exact-test).
In silico predictions for three different cancer genes, Retinoblastoma (Rb), TGF-beta-2 receptor (TGFBR2), and pleiomorphic adenoma gene 1 (PLAG1), were confirmed experimentally by in vitro assays. Using a luciferase reporter assay, three microRNAs tested (miR-106a, miR-20a and miR-26a-1) caused a significant reduction of protein translation relative to the scrambled control oligoRNAs in transfected MEG-01 cells (FIG. 6). Retinoblastoma 3′UTR, for example, was found to interact functionally with miR-106a. The biological significance of this miRNA:mRNA interaction is reinforced by previous reports showing that the Rb1 gene is normally transcribed in colon cancers, whilst various fractions of cells do not express Rb1 protein (Ali, A. A., et al., FASEB J. 7:931-937 (1993)). This finding suggests the existence of a post-transcriptional mechanism for regulating Rb1 that could be explained by concomitant miR-106a over-expression in colon carcinoma (FIG. 4). Furthermore, mir-20a is down-regulated in breast cancer (FIG. 4) and TFGBR2 protein is expressed in the epithelium of breast cancer cells (Buck, M. B., et al., Clin. Cancer Res. 10:491-498 (2004)). Conversely, the over-expression of mir-20a in colon cancer may represent a novel mechanism for down-regulating TGFBR2, in addition to mutational inactivation (Biswas, S., et al., Cancer Res. 64:687-692 (2004)).
Finally, a set of patient samples was tested to verify whether RB1 protein expression correlates with miR-106a expression (FIG. 5 and FIG. 6B). As expected, in gastric, prostate and lung tumor samples RB1 was down-regulated (in respect to the paired normal) and miR-106a was found to be over-expressed, while in breast tumor samples, where miR-106a is slightly down-regulated (FIG. 5 and FIG. 6B), RB1 is expressed at slightly higher levels then in the paired normal control.
These experimental proofs reinforce the hypothesis that key cancer genes are regulated by aberrant expression of miRs in solid cancers. These data add novel examples to the list of microRNA with important cancer gene targets, as previously shown by Johnsson et al. (Johnson, S. M., et al., Cell 120: 635-647 (2005)) for the let-7:Ras interaction, O'Donnell et al. (O'Donnell, K. A., et al., Nature 435:839-843 (2005)) for the miR-17-5p:cMyc interaction, and Cimmino et al. (Cimmino, A., et al., Proc. Natl. Acad. Sci. USA 102:13944-13949 (2005)) for the mir-16:Bc12 interaction. Notably, miR-17-5p and miR-16 are members of the miRNA solid cancer signature described herein.







TABLE 15







Oncogenes and tumor suppressor genes predicted by TargetScanS


as targets or microRNAs from the comprehensive cancer subset.*









miRNA gene
Gene Name
Gene description





miR-26a, miR-146
ABL2
v-abl Abelson murine leukemia viral oncogene




homolog 2 (arg, Abelson-related gene)


miR-107
AF5q31
ALL1 fused gene from 5q31


miR-20, miR-125b
AKT3
v-akt murine thymoma viral oncogene homolog 3


miR-26a, miR-155
APC
adenomatosis polyposis coli


miR-125b


miR-26a, miR-218
ARHGEF12
RHO guanine nucleotide exchange factor (GEF) 12




(LARG)


miR-107, miR-221
ARNT
aryl hydrocarbon receptor nuclear translocator


miR-192
ATF1
activating transcription factor 1


miR-26a
ATM
Ataxia telangiectasia mutated (includes




complementation groups A, C and D)


miR-24
AXL
AXL receptor tyrosine kinase


miR-26a, miR-107,
BCL11A
B-cell CLL/lymphoma 11A


miR-146, miR-155


miR-138, miR-92


miR-20
BCL11B
B-cell CLL/lymphoma 11B (CTIP2)


miR-21
BCL2
B-cell CLL/lymphoma 2


miR-26a, miR-26a
BCL6
B-cell CLL/lymphoma 6 (zinc finger protein 51)


miR-20,


miR-92
BCL9
B-cell CLL/lymphoma 9


miR-26a, miR-223
CBFB
core-binding factor, beta subunit


miR-221, miR-125b


miR-218
CCDC6
coiled-coil domain containing 6


miR-20
CCND1
cyclin D1 (PRAD1: parathyroid adenomatosis 1)


miR-26a, miR-20
CCND2
cyclin D2


miR-26a, miR-107,
CDK6
cyclin-dependent kinase 6


miR-92


miR-20
CDKN1A
cyclin-dependent kinase inhibitor 1A (p21, Cip1)


miR-221, miR-92
CDKN1C
cyclin-dependent kinase inhibitor 1C (p57, Kip2)


miR-24
CDX2
caudal type homeo box transcription factor 2


miR-92
CEBPA
CCAAT/enhancer binding protein (C/EBP), alpha


miR-26a
CLTC
clathrin, heavy polypeptide (Hc)


miR-218
COL1A1
collagen, type I, alpha 1


miR-26a
CREBBP
CREB binding protein (CBP)


miR-20
CRK
v-crk avian sarcoma virus CT10 oncogene




homolog


miR-20
CSF1
colony stimulating factor 1 (macrophage)


miR-221, miR-192
DDX6
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 6




(RNA helicase, 54 kD)


miR-138
DEK
DEK oncogene (DNA binding)


miR-20
E2F1
E2F transcription factor 1


miR-20
ELK3
ELK3, ETS-domain protein (SRF accessory protein 2)


miR-24
ELL
ELL gene (11-19 lysine-rich leukemia gene)


miR-26a, miR-138
ERBB4
v-erb-a avian erythroblastic leukemia viral




oncogene homolog-like 4


miR-221, miR-155,
ETS1
v-ets avian erythroblastosis virus E26 oncogene


miR-125b

homolog 1


miR-20
ETV1
ets variant gene 1


miR-125b
ETV6
ets variant gene 6 (TEL oncogene)


miR-223
FAT
FAT tumor suppressor (Drosophila) homolog


miR-223, miR-125b,
FGFR2
fibroblast growth factor receptor 2


miR-218


miR-92
FLI1
Friend leukemia virus integration 1


miR-24, miR-20
FLT1
fms-related tyrosine kinase 1 (vascular endothelial




growth factor/vascular permeability factor receptor)


miR-221
FOS
v-fos FBJ murine osteosarcoma viral oncogene




homolog


miR-92
FOXG1B
forkhead box G1B


miR-223
FOXO3A
forkhead box O3A


miR-125b
GOLGA5
golgi autoantigen, golgin subfamily a, 5 (PTC5)


miR-138
GPHN
gephyrin (GPH)


miR-107, miR-223,
HLF
hepatic leukemia factor


miR-20, miR-218


miR-26a, miR-107
HMGA1
high mobility group AT-hook 1


miR-20
HOXA13
homeo box A13


miR-92
HOXA9
homeo box A9


miR-125b
IRF4
interferon regulatory factor 4


miR-146, miR-20,
JAZF1
juxtaposed with another zinc finger gene 1


miR-138


miR-92
JUN
v-jun avian sarcoma virus 17 oncogene homolog


miR-155
KRAS
v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene




homolog


miR-218
LASP1
LIM and SH3 protein 1


miR-218
LHFP
lipoma HMGIC fusion partner


miR-125b, miR-218
LIFR
leukemia inhibitory factor receptor


miR-223
LMO2
LIM domain only 2 (rhombotin-like 1) (RBTN2)


miR-223, miR-155,
MAF
v-maf musculoaponeurotic fibrosarcoma (avian)


miR-125b, miR-92

oncogene homolog


miR-92
MAP2K4
mitogen-activated protein kinase kinase 4


miR-146, miR-20
MAP3K8
mitogen-activated protein kinase kinase kinase 8


miR-125b
MAX
MAX protein


miR-218
MCC
mutated in colorectal cancers


miR-24
MEN1
multiple endocrine neoplasia I


miR-138
MLLT6
myeloid/lymphoid or mixed-lineage leukemia




(trithorax homolog, Drosophila): translocated to, 6




(AF17)


miR-192
MSN
moesin


miR-24
MYB
v-myb avian myeloblastosis viral oncogene




homolog


miR-107, miR-223,
MYBL1
v-myb avian myeloblastosis viral oncogene


miR-146, miR-221,

homolog-like 1


miR-155, miR-218


miR-107, miR-20
MYCN
v-myc avian myelocytomatosis viral related




oncogene, neuroblastoma derived


miR-107, miR-92
MYH9
myosin. heavy polypeptide 9. non-muscle


miR-24
MYST4
MYST histone acetyltransferase (monocytic




leukemia) 4 (MORF)


miR-20
NBL1
neuroblastoma, suppression of tumorigenicity 1


miR-125b
NIN
ninein (GSK3B interacting protein)


miR-26a, miR-107
NKTR
natural killer-tumor recognition sequence


miR-92
NOTCH1
Notch homolog 1, translocation-associated




(Drosophila) (TAN1)


miR-24
NTRK3
neurotrophic tyrosine kinase, receptor, type 3


miR-125b
PCSK7
proprotein convertase subtilisin/kexin type 7


miR-24, miR-146
PER1
period homolog 1 (Drosophila)


miR-146, miR-125b,
PHOX2B
paired-like homeobox 2b


miR-138,


miR-155
PICALM
phosphatidylinositol binding clathrin assembly




protein (CALM)


miR-24, miR-26a
PIM1
pim-1 oncogene


miR-24, miR-26a,
PLAG1
pleiomorphic adenoma gene 1


miR-21, miR-107,


miR-20, miR-155


miR-218
RAB8A
RAB8A, member RAS oncogene family


miR-24, miR-221
RALA
v-ral simian leukemia viral oncogene homolog A




(ras related)


miR-138
RARA
retinoic acid receptor, alpha


miR-20, miR-192
RB1
retinoblastoma 1 (including osteosarcoma)


miR-20,
RBL1
retinoblastoma-like 1 (p107)


miR-20
RBL2
retinoblastoma-like 2 (p130)


miR-155, miR-138
REL
v-rel avian reticuloendotheliosis viral oncogene




homolog


miR-20, miR-138
RHOC
ras homolog gene family, member C


miR-20, miR-192
RUNX1
runt-related transcription factor 1 (AML1)


miR-107, miR-223
SEPT6
seplin 6


miR-146, miR-20,
SET
SET translocation


miR-125b


miR-21, miR-20,
SKI
v-ski avian sarcoma viral oncogene homolog


miR-155, miR-218


miR-26a, miR-146
SMAD4
SMAD, mothers against DPP homolog 4




(Drosophila)


miR-155
SPI1
spleen focus forming virus (SFFV) proviral




integration oncogene spi1


miR-125b
SS18
synovial sarcoma translocation, chromosome 18


miR-107, miR-155
SUFU
suppressor of fused homolog (Drosophila)


miR-92
TAF15
TAF15 RNA polymerase II, TATA box binding




protein (TBP)-associated factor, 68 kDa


miR-26a, miR-221,
TCF12
transcription factor 12 (HTF4, helix-loop-helix


miR-138

transcription factors 4)


miR-21, miR-20
TGFBR2
transforming growth factor, beta receptor II




(70-80 kD)


miR-24, miR-26a,
TOP1
topoisomerase (DNA) I


miR-92


miR-138
TPM4
tropomyosin 4


miR-20
TRIP11
thyroid hormone receptor interactor 11


miR-92
TSC1
Tuberous sclerosis 1


miR-20
TSG101
Tumor susceptibility gene 101


miR-20
TUSC2
Tumor suppressor candidate 2


miR-24
VAV1
vav 1 oncogene


miR-125b
VAV2
vav 2 oncogene


miR-107
WHSC1
Wolf-Hirschhorn syndrome candidate 1(MMSET)


miR-138
WHSC1L1
Wolf-Hirschhorn syndrome candidate 1-like 1




(NSD3)


miR-26a
WNT5A
wingless-type MMTV integration site family,




member 5A


miR-26a, miR-20,
YES1
v-yes-1 Yamaguchi sarcoma viral oncogene


miR-125b

homolog 1


miR-107, miR-221
ZNF198
zinc finger protein 198


miR-218
ZNFN1A1
zinc finger protein, subfamily 1A, 1 (Ikaros)





*Known cancer genes (e.g., tumor suppressors, oncogenes) comprise those identified in the Cancer Gene Census at www.sanger.ac.uk/genetics/CGP/Census/ or reported by OMIM at www.ncbi.nlm.nih.gov.






The relevant teachings of all publications cited herein that have not explicitly been incorporated by reference, are incorporated herein by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method of diagnosing a subject having stomach cancer, comprising:
obtaining a nucleic acid-containing test sample from a subject suspected of having stomach cancer, wherein the sample is extracted from stomach cells;
measuring, by hybridization assay, a level of at least one miR gene product of miR-21 in the test sample;
comparing the level of at least one miR gene product of miR-21 in the test sample from the subject to a control level of non-cancerous stomach cells of at least one corresponding miR gene product of miR-21; and
diagnosing the subject as having stomach cancer, if the level of the at least one miR gene product in the test sample from the subject is miR-21 and is greater than the control level for the corresponding miR gene product of miR-21.

2. A method of diagnosing a subject having stomach cancer, comprising:
a.) reverse transcribing at least one miR gene product of miR-21 from a nucleic acid-containing test sample from stomach tissue of the subject to provide at least one corresponding miR gene product target oligonucleotides;
b.) hybridizing the at least one corresponding miR gene product target oligodeoxynucleotides to a microarray comprising miRNA-specific probe oligonucleotides that include at least one corresponding miRNA-specific probe oligonucleotides, specific for miR-21, to provide a miR-21 hybridization profile for the test sample;
c.) comparing the test sample miR-21 hybridization profile to at least one miR-21 control hybridization profile of normal stomach tissue; and
d.) diagnosing the subject as having stomach cancer if the level of at least one miR-21 gene product in the test sample is greater than the control level of corresponding miR-21 gene product.

3. A method of diagnosing a subject having stomach cancer, comprising:
measuring, by hybridization assay, the level of at least miR-21 gene product in a test sample from stomach cells of a subject;
comparing the measured level of at least miR-21 gene product in a test sample from a subject to a control level of non-cancerous stomach cells for at least one miR-21 gene product; and
diagnosing the subject as having stomach cancer, if the level of at least miR-21 gene product in the test sample from the subject is greater than the control level of miR-21 gene product.

4. A method of diagnosing a subject having stomach cancer, comprising:
a.) reverse transcribing at least miR-21 gene product from a test sample from stomach cells of a subject to provide at least one corresponding miR-21 gene product target oligonucleotide;
b.) hybridizing the at least one miR-21 gene product target oligonucleotide to a microarray comprising miRNA-specific probe oligonucleotides that include at least one corresponding miR-21 RNA specific probe oligonucleotide to provide a miR-21 hybridization profile for the test sample;
c.) comparing the test sample miR-21 hybridization profile to at least one control miR-21 hybridization profile, wherein the control is derived from miRNA expression of non-cancerous stomach cells; and
d.) diagnosing the subject as having stomach cancer if the level of at least one miR-21 gene product in the hybridization profile from the test sample is greater than the level of corresponding miR-21 gene product in the hybridization profile from the control miR-21 hybridization profile.

5. A method for detecting likelihood of stomach cancer in a subject, the method comprising:
detecting one or both of the presence and amount of one or more biomarkers in a biological sample comprised of stomach cells from the subject, wherein at least one of the biomarkers is a miR-21 gene product; and
classifying the subject as likely to have stomach cancer if the amount of the miR-21 gene product is increased in the biological sample as compared to a non-stomach cancer reference amount of a corresponding miR-21 gene product biomarker.

6. The method of claim 1, wherein the subject is a human.

7. A method of facilitating a diagnosis of a stomach cancer in a subject, comprising:
obtaining a test sample from gastric cells of the subject;
measuring a level of expression of; and
comparing the level of miR-21 with a corresponding pre-determined level for miR-21, the pre-determined level being obtained from a control sample from a control subject who has stomach cancer, wherein having a level of miR-21 that is at or above the corresponding predetermined level indicates that the subject has stomach cancer.