MicroRNAs in colorectal cancer: from diagnosis to targeted therapy.

Colorectal cancer (CRC) is one of the major healthcare problems worldwide and its processes of genesis include a sequence of molecular pathways from adenoma to carcinoma. The discovery of microRNAs, a subset of regulatory non-coding RNAs, has added new insights into CRC diagnosis and management. Together with several causes of colorectal neoplasia, aberrant expression of oncomiRs (oncogenic and tumor suppressor miRNAs) in cancer cells was found to be indirectly result in up- or down-regulation of targeted mRNAs specific to tumor promoter or inhibitor genes. The study of miRNAs as CRC biomarkers utilizes expression profiling methods from traditional tissue samples along with newly introduced non-invasive samples of faeces and body fluids. In addition, miRNAs could be employed to predict chemo- and radio-therapy responses and be manipulated in order to alleviate CRC characteristics. The scope of this article is to provide a comprehensive review of scientific literature describing aberrantly expressed miRNAs, and consequently dysregulation of targeted mRNAs along with the potential role of miRNAs in CRC diagnosis and prognosis, as well as to summarize the recent findings on miRNA-based manipulation methods with the aim of advancing in anti-CRC therapies.

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title: Increased burden of colorectal cancer in Asia.
abstract: The incidence and mortality of colorectal cancer (CRC) is rising rapidly in Asia. It seems that ethnicity has an important etiological role in CRC in Asia. However the incidence, anatomical distribution and mortality of CRC among Asian populations are not different from those in Western countries. There is little support by health authorities for CRC screening and very low public awareness of this emerging epidemic in Asia. The increasing rate of CRC in Asia means that we need to take action immediately to prevent CRC and to diagnose the disease at the early stages by introducing CRC screening in countries at high risk of an increasing burden of CRC.

title: Knowledge and Practice of Iranians Toward Colorectal Cancer, and Barriers to Screening
abstract: Colorectal cancer (CRC) is the third most common malignancy in Iran. Limited data are available on knowledge and barriers in regard to CRC and screening tests in Iran. The aim of the study was to characterize knowledge, practice, and barriers toward CRC and its screening tests among an Iranian at-risk population.  This cross-sectional study was conducted with participation of 200 individuals of both genders aged 50 years or older in a teaching hospital in Tehran, Iran. Data were collected via face-to-face interviews. A questionnaire containing demographics; knowledge about CRC and screening tests; screening practice; and reasons for not being screened was administered. The reliability alpha for knowledge items was 0.52.  The age of the participants ranged from 50 to 83 years (mean 60.13). Overall, 11% of the respondents reported prior screening by either fecal occult blood test (6.5%) or colonoscopy (4.5%). The majority of individuals had poor knowledge although respondents with prior screening obtained slightly higher score in comparison with nonparticipants in screening (26.74 vs. 23.24; P<0.05). Four commonly cited reasons for not having CRC tests were “doctor did not recommend the test,” “did not think it was needed,” “never think of the test,” and “no symptoms/problems” which were reported by 29%, 26%, 20%, and 17% of the participants, respectively.  It is necessary to design appropriate educational interventions to increase the general population's knowledge about CRC and screening before implementing preventive programs in Iran.

title: Non-coding RNAs: The cancer X factor
abstract: Eda Yildirim, Jeannie Lee and colleagues present intriguing evidence that the long non-coding RNA X-inactive specific transcript (Xist) is required for maintaining X-chromosome inactivation, and that the loss of Xist after X inactivation can cause haematopoietic cancers in mice.

title: A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans
abstract: How left/right functional asymmetry is layered on top of an anatomically symmetrical nervous system is poorly understood. In the nematode Caenorhabditis elegans, two morphologically bilateral taste receptor neurons, ASE left (ASEL) and ASE right (ASER), display a left/right asymmetrical expression pattern of putative chemoreceptor genes that correlates with a diversification of chemosensory specificities. Here we show that a previously undefined microRNA termed lsy-6 controls this neuronal left/right asymmetry of chemosensory receptor expression. lsy-6 mutants that we retrieved from a genetic screen for defects in neuronal left/right asymmetry display a loss of the ASEL-specific chemoreceptor expression profile with a concomitant gain of the ASER-specific profile. A lsy-6 reporter gene construct is expressed in less than ten neurons including ASEL, but not ASER. lsy-6 exerts its effects on ASEL through repression of cog-1, an Nkx-type homeobox gene, which contains a lsy-6 complementary site in its 3′ untranslated region and that has been shown to control ASE-specific chemoreceptor expression profiles. lsy-6 is the first microRNA to our knowledge with a role in neuronal patterning, providing new insights into left/right axis formation.

title: Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis
abstract: Gradients of signalling and transcription factors govern many aspects of embryogenesis, highlighting the need for spatiotemporal control of regulatory protein levels. MicroRNAs are phylogenetically conserved small RNAs that regulate the translation of target messenger RNAs, providing a mechanism for protein dose regulation. Here we show that microRNA-1-1 (miR-1-1) and miR-1-2 are specifically expressed in cardiac and skeletal muscle precursor cells. We found that the miR-1 genes are direct transcriptional targets of muscle differentiation regulators including serum response factor, MyoD and Mef2. Correspondingly, excess miR-1 in the developing heart leads to a decreased pool of proliferating ventricular cardiomyocytes. Using a new algorithm for microRNA target identification that incorporates features of RNA structure and target accessibility, we show that Hand2, a transcription factor that promotes ventricular cardiomyocyte expansion, is a target of miR-1. This work suggests that miR-1 genes titrate the effects of critical cardiac regulatory proteins to control the balance between differentiation and proliferation during cardiogenesis.

title: Stem cell division is regulated by the microRNA pathway
abstract: One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

title: The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation
abstract: Deciphering the mechanisms underlying skeletal muscle-cell differentiation in mammals is an important challenge. Cell differentiation involves complex pathways regulated at both transcriptional and post-transcriptional levels. Recent observations have revealed the importance of small (20–25 base pair) non-coding RNAs (microRNAs or miRNAs) that are expressed in both lower organisms and in mammals. miRNAs modulate gene expression by affecting mRNA translation or stability. In lower organisms, miRNAs are essential for cell differentiation during development; some miRNAs are involved in maintenance of the differentiated state. Here, we show that miR-181, a microRNA that is strongly upregulated during differentiation, participates in establishing the muscle phenotype. Moreover, our results suggest that miR-181 downregulates the homeobox protein Hox-A11 (a repressor of the differentiation process), thus establishing a functional link between miR-181 and the complex process of mammalian skeletal-muscle differentiation. Therefore, miRNAs can be involved in the establishment of a differentiated phenotype — even when they are not expressed in the corresponding fully differentiated tissue.

title: RNAi: The Nuts and Bolts of the RISC Machine
abstract: Research on RNA interference and microRNA pathways continues to accelerate. Combinations of biochemical, genetic, and structural approaches are providing new insights into the mechanism by which small, approximately 21 nt long RNAs find their way into the RISC effector complexes and how the RISCs execute their mission of RNA-guided posttranscriptional gene silencing.

title: A pancreatic islet-specific microRNA regulates insulin secretion
abstract: MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression. Several miRNAs in animals exhibit tissue-specific or developmental-stage-specific expression, indicating that they could play important roles in many biological processes. To study the role of miRNAs in pancreatic endocrine cells we cloned and identified a novel, evolutionarily conserved and islet-specific miRNA (miR-375). Here we show that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion. The mechanism by which secretion is modified by miR-375 is independent of changes in glucose metabolism or intracellular Ca2+-signalling but correlated with a direct effect on insulin exocytosis. Myotrophin (Mtpn) was predicted to be and validated as a target of miR-375. Inhibition of Mtpn by small interfering (si)RNA mimicked the effects of miR-375 on glucose-stimulated insulin secretion and exocytosis. Thus, miR-375 is a regulator of insulin secretion and may thereby constitute a novel pharmacological target for the treatment of diabetes.

title: RNAi and miRNA in viral infections and cancers.
abstract: Since the first report of RNA interference (RNAi) less than a decade ago, this type of molecular intervention has been introduced to repress gene expression in vitro and also for in vivo studies in mammals. Understanding the mechanisms of action of synthetic small interfering RNAs (siRNAs) underlies use as therapeutic agents in the areas of cancer and viral infection. Recent studies have also promoted different theories about cell-specific targeting of siRNAs. Design and delivery strategies for successful treatment of human diseases are becomingmore established and relationships between miRNA and RNAi pathways have been revealed as virus-host cell interactions. Although both are well conserved in plants, invertebrates and mammals, there is also variabilityand a more complete understanding of differences will be needed for optimal application. RNA interference (RNAi) is rapid, cheap and selective in complex biological systems and has created new insight sin fields of cancer research, genetic disorders, virology and drug design. Our knowledge about the role of miRNAs and siRNAs pathways in virus-host cell interactions in virus infected cells is incomplete. There are different viral diseases but few antiviral drugs are available. For example, acyclovir for herpes viruses, alpha-interferon for hepatitis C and B viruses and anti-retroviral for HIV are accessible. Also cancer is obviously an important target for siRNA-based therapies, but the main problem in cancer therapy is targeting metastatic cells which spread from the original tumor. There are also other possible reservations and problems that might delay or even hinder siRNA-based therapies for the treatment of certain conditions; however, this remains the most promising approach for a wide range of diseases. Clearly, more studies must be done to allow efficient delivery and better understanding of unwanted side effects of siRNA-based therapies. In this review miRNA and RNAi biology, experimental design, anti-viral and anti-cancer effects are discussed.

title: Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue.
abstract: By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase followed by TaqMan PCR expression analysis on stool and tissue samples using fifteen human (Homo sapiens, hsa) micro(mi)RNA genes selected by careful analysis of the peer-reviewed literature, we were able to monitor changes at various stages of CRC, allowing for reliable diagnostic screening of colon cancer particularly at the early, pre-malignant stages, and for difficult-to-treat active ulcerative colitis (UC). Although the expression of some of the miRNA genes tested in tissue showed less variability in CRC or UC patients than in stool, the stool by itself appears well-suited to screening. A miRNA approach using stool samples promises to offer more sensitivity and specificity than currently used screening genomic, methylomic or proteomic methods for colon cancer. Larger prospective clinical studies utilizing stool derived from many control, colon cancer or UC patients, to allow for a statistically valid analysis, are now urgently required to standardize test performance and determine the true sensitivity and specificity of the miRNA screening approach, and to provide a numerical underpinning for these diseases as a function of total RNA. Moreover, when a miRNA screening test is combined with analysis of a messenger(m)RNA expression test, which has also been considered in earlier studies to be a highly sensitive and a very specific and reliable transcriptomic approach, as outlined in this article, bioinformatics can be used to correlate microRNA seed data with mRNA target data in order to gain a mechanistic understanding of how miRNAs regulate gene expression, enabling understanding of why these miRNA genes should be informative in a screening test.

title: Epigenetic regulation of microRNA expression in colorectal cancer
abstract: In the last years, microRNAs (miRNA) have emerged as new molecular players involved in carcinogenesis. Deregulation of miRNAs expression has been shown in different human cancer but the molecular mechanism underlying the alteration of miRNA expression is unknown. To identify tumor‐supressor miRNAs silenced through aberrant epigenetic events in colorectal cancer (CRC), we used a sequential approach. We first identified 5 miRNAs down‐regulated in patient with colorectal cancer samples and located around/on a CpG island. Treatment with a DNA methyltransferase inhibitor and a HDAC inhibitor restored expression of 3 of the 5 microRNAs (hsa‐miR‐9, ) in 3 CRC cell lines. Expression of hsa‐miR‐9 was inversely correlated with methylation of their promoter regions as measure by MSP and bisulphate sequencing. Further, methylation of the hsa‐miR‐9‐1, hsa‐miR‐129‐2 and  CpG islands were frequently observed in CRC cell lines and in primary CRC tumors, but not in normal colonic mucosa. Finally, methylation of hsa‐miR‐9‐1 was associated with the presence of lymph node metastasis. In summary, our results aid in the understanding of miRNA gene regulation showing that aberrant DNA methylation and histone modifications work together to induce silencing of miRNAs in CRC.

title: Oncomirs — microRNAs with a role in cancer
abstract: MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators. They regulate diverse biological processes, and bioinformatic data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumour suppressors and oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer.

title: The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis
abstract: MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes. Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently and still remains largely unexplored. To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay. We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently. Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44. We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs.

title: microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN
abstract: Gastric cancer is one of the most common carcinomas in China. microRNAs, a type of non-coding RNA, are important specific regulators and are involved in numerous bioprocesses of an organism. microRNA-21 (miR-21) has been identified as the most suitable choice for further investigation because it is overexpressed in nearly all solid tumors; furthermore, it has been demonstrated that miR-21 is involved in the genesis and progression of human cancer. It has been reported that PTEN, an important tumour suppressor, is regulated by multiple miRNAs. Thus, in this study we focused on the expression and significance of miR-21 in gastric cancer tissues, and the role of miR-21 in the biological behaviour and the expression of PTEN in gastric cancer cells. Real-time PCR was used to detect miR-21 expression in gastric cancer tissues, the adjacent normal tissues, and the gastric cell lines. The gastric cancer cell line BGC-823 was transfected with pre-miR-21/miR-21 inhibitor to overexpress/downregulate miR-21. The influence of miR-21 on the biological behaviour of gastric cancer cells was evaluated using the CCK-8 kit, FCMs, the scratch healing assay and the transwell test. Western blotting and the Luciferase Reporter Assay were used to evaluate the change of PTEN expression after lowered expression of miR-21 in gastric cancer cell lines. Real-time PCR analysis indicated that miR-21 exhibited higher expression in gastric cancer tissues compared to the adjacent non-tumor tissues. miR-21 expression was significantly associated with the degree of differentiation of the tumour tissues (P=0.004), as well as local invasion and lymph node metastasis (P<0.01). After transfection, pre-miR21 BGC-823 cells grew faster than the negative and control groups (P<0.01). The reduction in miR-21 expression demonstrated a remarkable effect on the biological behaviour of gastric cancer cells (P<0.05); the pre-miR-21-transfected cells healed more quickly compared to the control cells in the scratch healing assay, whereas the transwell test indicated that cell migration in vitro was notably inhibited with the downregulation of miR-21 (P<0.05). The western blot results and Luciferase Reporter Assay demonstrated that PTEN expression was remarkably increased after miR-21 inhibition (P<0.05). microRNA-21 expression was upregulated in gastric carcinoma tissues and was significantly associated with the degree of differentiation of tumour tissues, local invasion and lymph node metastasis. Overexpression of miR-21 promoted BGC-823 cell growth, invasion and cell migration in vitro, whereas downregulation of miR-21 exhibited a stronger inhibitory effect on the biological behaviour of gastric cancer cells; additionally, miR-21 inhibition may upregulate the PTEN expression level, which indicates that PTEN may be a target gene for gastric cancer initiation and development.

title: MicroRNAs and colon and rectal cancer: Differential expression by tumor location and subtype
abstract: MicroRNAs are thought to have an impact on cell proliferation, apoptosis, stress responses, maintenance of stem cell potency, and metabolism and are, therefore, important in the carcinogenic process. In this study, we examined 40 colon tumors, 30 rectal tumors, and 30 normal tissue samples (10 proximal colon, 10 distal colon, and 10 rectal paired with cancer cases) to examine miRNA expression profiles in colon and rectal tumors. MiRNA expression levels were adjusted for multiple comparisons; tumor tissue was compared with noncancerous tissue from the same site. A comparison of normal tissue showed 287 unique miRNAs that were significantly differentially expressed at the 1.5‐fold level and 73 with over a two‐fold difference in expression between colon and rectal tissue. Examination of miRNAs that were significantly differentially expressed at the 1.5‐fold level by tumor phenotype showed 143 unique miRNAs differentially expression for microsatellite instability positive (MSI+) colon tumors; 129 unique miRNAs differentially expressed for CpG Island Methylator Phenotype positive (CIMP+) colon tumors; 135 miRNAs were differentially expressed for KRAS2‐mutated colon tumors, and 139 miRNAs were differentially expressed for TP53‐mutated colon tumors. Similar numbers of differentially expressed miRNAs were observed for rectal tumors, although the miRNAs differentially expressed differed. There were 129 unique miRNAs for CIMP+, 143 unique miRNAs for KRAS2‐mutated, and 136 unique miRNAs for TP53‐mutated rectal tumors. These results suggest the importance of miRNAs in colorectal cancer and the need for studies that can confirm these results and provide insight into the diet, lifestyle, and genetic factors that influence miRNA expression.

title: Association of microRNA expression with microsatellite instability status in colorectal adenocarcinoma.
abstract: MicroRNAs (miRNA), small noncoding RNAs, are potential diagnostic and prognostic markers, as well as therapeutic targets. miRNA profiles of colorectal carcinomas have not been studied extensively in the context of microsatellite instability (MSI) status. We therefore evaluated 55 paired colorectal adenocarcinomas (CRC) and non-neoplastic mucosa samples using a panel of 24 miRNAs selected by literature review and prior studies in our laboratory. Stem-loop reverse transcriptase quantitative (real-time) polymerase chain reaction assays were done on RNA extracted from formalin-fixed, paraffin-embedded tissue of resection specimens. When miRNA expression was compared with clinicopathologic features and MSI status, eleven miRNAs (miR-183, -31, -20, -25, -92, -93, -17, -135a, -203, -133b, and -223) were over-expressed in CRC relative to mucosa, and nine (miR-192, -215, -26b, -143, -145, -191, -196a, -16, and let-7a) were under-expressed in CRC. Relative expression of miR-92, -223, -155, -196a, -31, and -26b were significantly different among MSI subgroups, and miR-31 and miR-223 were overexpressed in CRC of patients with hereditary non-polyposis colorectal cancer syndrome (Lynch syndrome). Our findings indicate that miRNA expression in CRC is associated with MSI subgroups, including low MSI and HNPCC-associated cancers, and that miRNAs may have posttranscriptional gene regulatory roles in these MSI subgroups and possible effects on the clinicopathologic and biomarker characteristics.

title: Frequent MSI mononucleotide markers for diagnosis of hereditary nonpolyposis colorectal cancer.
abstract: Failure in the DNA mismatch repair system is commonly accompanied by microsatellite instability and leads to colorectal cancer. The aim of this study was to find the most frequent of five mononucleotide markers in order to devise the simplest diagnostic strategy for identification of patients with hereditary nonpolyposis colorectal cancer (HNPCC) who were defined by defects in mismatch repair system.



78 patients with colorectal cancer were recruited for this investigation. Five mononucleotide markers, NR-27, NR-21, NR-24, BAT-25 and BAT-26, were used as a pentaplex panel to determine MSI status.



Two out of five mononucleotide markers, NR-21 (25.6%) and BAT-25 (23.1%) showed more instability than the others.



In defining individuals with colorectal cancer, BAT25 and NR-21 may provide diagnostic assistance.

title: Simplified MSI marker panel for diagnosis of colorectal cancer.
abstract: Colorectal cancers (CRCs) tumors are diagnosed by microsatellite instability (MSI) due to accumulation of insertion/deletion mutations in tandem repeats of short DNA motifs (1-6 bp) called microsatellites. Microsatellite instability (MSI) is not only a hallmark marker for screening of hereditary nonpolyposis colorectal cancer (HNPCC), but also a prognostic and predictive marker for sporadic colorectal cancer. Our objective was to determine and study of five mononucleotide microsatellite markers status among Iranian patients with HNPCC and sporadic colorectal cancer.



In the current investigation 80 sporadic CRC and 80 HNPCC patients were evaluated for MSI. The pentaplex panel including 5 quasimonomorphic mononucleotide repeats (NR-21, BAT-26, BAT-25, NR-27 and NR-24) was used.



Our findings showed that the NR-21 was the most frequent instable marker among the other markers. 53% and 25.6% specimens had instability in sporadic CRC and HNPCC, respectively. Furthermore, the frequencies of instability BAT-25 was determined in 20% sporadic CRC and 23% HNPCC samples. Interestingly our results demonstrated that the frequency of instability NR-24 was similar 20% sporadic CRC and 20.5% HNPCC. Moreover, percentage of NR-27 in HNPCC was 19.2 and 0% in sporadic CRC. Finally, BAT-26 was instable in 21.8% HNPCC patients while we could find 6.6% instability for BAT-26 in sporadic cases.



It seems that among 5 mononucleotides markers NR-21 was the most useful marker for diagnosis HNPCC and sporadic cancer. Following NR-21, BAT-25 and NR-24 are the most reliable markers. Therefore using a triplex panel including 3 aforementioned MSI markers should be more promising markers for identifying MSI status in both patients with HNPCC and/or sporadic colorectal cancer.

title: Limited diagnostic value of microRNAs for detecting colorectal cancer: a meta-analysis.
abstract: MicroRNAs have been demonstrated to play important roles in the development and progression of colorectal cancer. Several studies utilizing microRNAs as diagnostic biomarkers for colorectal cancer (CRC) have been reported. The aim of this meta-analysis was to comprehensively and quantitatively summarize the diagnostic value of microRNAs for detecting colorectal cancer.



We searched PubMed, Embase and Cochrane Library for published studies that used microRNAs as biomarkers for the diagnosis of colorectal cancer. Summary estimates for sensitivity, specificity and other measures of accuracy of microRNAs in the diagnosis of colorectal cancer were calculated using the bivariate random effects model. A summary receiver operating characteristic (SROC) curve was also generated to summarize the overall effectiveness of the test.



Thirteen studies from twelve published articles met the inclusion criteria and were included. The overall sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic odd ratio of microRNAs for the diagnosis of colorectal cancer were 0.81 (95%CI: 0.79-0.84), 0.78 (95%CI: 0.75-0.82), 4.14 (95%CI: 2.90- 5.92), 0.24 (95%CI: 0.19-0.30), and 19.2 (95%CI: 11.7-31.5), respectively. The area under the SROC curve was 0.89.



The current evidence suggests that the microRNAs test might not be used alone as a screening tool for CRC. Combining microRNAs testing with other conventional tests such as FOBT may improve the diagnostic accuracy for detecting CRC.

title: A genetic variant in MiR-146a modifies digestive system cancer risk: a meta-analysis.
abstract: MicroRNAs (miRNAs) negatively regulate gene expression and act as tumor suppressors or oncogenes in oncogenesis. The association between a single nucleotide polymorphism (SNP) in miR-146a rs2910164 and susceptibility to digestive system cancers was inconsistent in previous studies. In this study, we conducted a literature search of PubMed to identify all relevant studies published before August 31, 2013. A total of 21 independent case-control studies were included in this updated meta-analysis with 9,558 cases and 10,614 controls. We found that the miR-146a rs2910164 polymorphism was significantly associated with decreased risk of digestive system cancers in an allele model (OR=0.90, 95%CI 0.87-0.94), homozygote model (OR=0.84, 95%CI 0.77-0.91), dominant model (OR=0.90, 95%CI 0.84-0.96), and recessive model (OR=0.85, 95%CI 0.79-0.91), while in a heterozygous model (OR = 0.99, 95% CI 0.89-1.11) the association showed marginal significance. Subgroup analysis by cancer site revealed decreased risk in colorectal cancer above allele model (OR=0.90, 95%CI 0.83- 0.97) and homozygote model (OR=0.85, 95%CI 0.72-1.00). Similarly, decreased cancer risk was observed when compared with allele model (OR=0.87, 95%CI 0.81-0.93) and recessive model (OR=0.81, 95%CI 0.72-0.90) in gastric cancer. When stratified by ethnicity, genotyping methods and quality score, decreased cancer risks were also observed. This current meta-analysis indicated that miR-146a rs2910164 polymorphism may decrease the susceptibility to digestive system cancers, especially in Asian populations.

title: MicroRNA profiling of clear cell renal cell carcinoma by whole‐genome small RNA deep sequencing of paired frozen and formalin‐fixed, paraffin‐embedded tissue specimens
abstract: Renal cell carcinoma (RCC) is one of the leading causes of cancer mortality. Characterization of microRNA (miRNA) expression of RCC will help disclose new pathogenic pathways in tumourigenesis and progression and may lead to the development of molecular biomarkers and target‐specific therapies for diagnosis, prognostication and treatment. With limitations in test specificity and the ability to detect novel miRNA and other small non‐coding RNAs (smRNAs), microarray and RT–PCR techniques are being replaced by the evolving deep‐sequencing technologies, at least in the discovery phase. Until now, cancer miRNA profiling of human benign and tumour specimen sets, using smRNA deep‐sequencing (smRNA‐seq), has not been reported. Specifically, due to concern over possible poor RNA quality/integrity, formalin‐fixed paraffin‐embedded (FFPE) samples have not been used for such studies. Here, we performed whole‐genome smRNA‐seq analysis using a benign and RCC specimen set and have successfully profiled the miRNA expression. Studies performed on paired frozen and FFPE specimens showed very similar results. Moreover, a comparison study of microarray, deep‐sequencing and RT–PCR methodologies also showed a high correlation among the three technologies. To our knowledge, this is the first study to demonstrate that FFPE specimens can be used reliably for miRNA deep‐sequencing analysis, making future large‐scale clinical cohort/trial‐based studies possible.

title: Robust Gene Expression Signature from Formalin-Fixed Paraffin-Embedded Samples Predicts Prognosis of Non–Small-Cell Lung Cancer Patients
abstract: The requirement of frozen tissues for microarray experiments limits the clinical usage of genome-wide expression profiling by using microarray technology. The goal of this study is to test the feasibility of developing lung cancer prognosis gene signatures by using genome-wide expression profiling of formalin-fixed paraffin-embedded (FFPE) samples, which are widely available and provide a valuable rich source for studying the association of molecular changes in cancer and associated clinical outcomes.  We randomly selected 100 Non–Small-Cell lung cancer (NSCLC) FFPE samples with annotated clinical information from the UT-Lung SPORE Tissue Bank. We microdissected tumor area from FFPE specimens and used Affymetrix U133 plus 2.0 arrays to attain gene expression data. After strict quality control and analysis procedures, a supervised principal component analysis was used to develop a robust prognosis signature for NSCLC. Three independent published microarray datasets were used to validate the prognosis model.  This study showed that the robust gene signature derived from genome-wide expression profiling of FFPE samples is strongly associated with lung cancer clinical outcomes and can be used to refine the prognosis for stage I lung cancer patients, and the prognostic signature is independent of clinical variables. This signature was validated in several independent studies and was refined to a 59-gene lung cancer prognosis signature.  We conclude that genome-wide profiling of FFPE lung cancer samples can identify a set of genes whose expression level provides prognostic information across different platforms and studies, which will allow its application in clinical settings.

title: Next Generation Sequencing: Potential and Application in Drug Discovery
abstract: The world has now entered into a new era of genomics because of the continued advancements in the next generation high throughput sequencing technologies, which includes sequencing by synthesis-fluorescent in situ sequencing (FISSEQ), pyrosequencing, sequencing by ligation using polony amplification, supported oligonucleotide detection (SOLiD), sequencing by hybridization along with sequencing by ligation, and nanopore technology. Great impacts of these methods can be seen for solving the genome related problems of plant and animal kingdom that will open the door of a new era of genomics. This may ultimately overcome the Sanger sequencing that ruled for 30 years. NGS is expected to advance and make the drug discovery process more rapid.

title: Reduced accumulation of specific microRNAs in colorectal neoplasia.
abstract: Short non-coding RNAs are known to regulate cellular processes including development, heterochromatin formation, and genomic stability in eukaryotes. Given the impact of these processes on cellular identity, a study was undertaken to investigate possible changes in microRNA (miRNA) levels during tumorigenesis. A total of 28 different miRNA sequences was identified in a colonic adenocarcinoma and normal mucosa, including 3 novel sequences and a further 7 that had previously been cloned only from mice. Human homologues of murine miRNA sequences, miR-143 and miR-145, consistently display reduced steady-state levels of the mature miRNA at the adenomatous and cancer stages of colorectal neoplasia.

title: MicroRNAs 143 and 145 are possible common onco-microRNAs in human cancers.
abstract: MicroRNAs (miRNAs) are endogenously expressed RNAs 18-24 nucleotides in length that regulate gene expression through translational repression by binding to a target mRNA. It is thought that the expression level of miRNAs, which act like antioncogenes, is frequently reduced in cancers because of chromosome deletion and epigenetic changes. Since the processing of miRNAs has been characterized to be enzymatic in nature, the expression levels of miRNAs are closely associated with the activity and levels of such enzymes. Here, we found that miRNA 143 and 145 expression levels were extremely reduced in colon cancer cells and commonly in the other kinds of cancer cells tested. The transfection of each precursor miRNA into the cells demonstrated a significant growth inhibition in human colon cancer DLD-1 and SW480 cells, and ERK5 was determined to be the target gene of miRNA 143. Since the presence of genomic loci of the miRNAs was confirmed by PCR in the cell lines and the precursor miRNAs exhibited a growth inhibitory effect in DLD-1 and SW480 cells, the early processes such as transcription and enzymatic modification from primary miRNAs to precursor miRNAs seemed to be commonly disturbed. These findings indicate that the miRNAs 143 and 145 could become good tumor markers and provide an important clue in the study of the mechanism of oncogenesis involving miRNAs.

title: let-7 microRNA functions as a potential growth suppressor in human colon cancer cells.
abstract: MicroRNAs (miRNAs) are endogenously expressed RNAs, 18-25 nucleotides in length, that repress protein translation through binding to target mRNAs. miRNAs have been implicated in many cellular processes including cell proliferation, differentiation, and death. Recently, let-7 miRNAs were found to regulate human RAS oncogene expression and to be often down-regulated in human lung tumors. In this study, we examined the expression of let-7 miRNAs in human colon cancer tumors and cell lines, with the result that 2 of 6 cases and 1 of 3 cell lines showed reduced expression of let-7. When let-7 low-expressing DLD-1 human colon cancer cells were transfected with let-7a-1 precursor miRNA, which is located at chromosome 9q22.3, the cells underwent significant growth suppression. At that time, the levels of RAS and c-myc proteins were lowered after the transfection, whereas the levels of both of their mRNAs remained almost unchanged. These findings suggest the involvement of let-7 miRNA in the growth of colon cancer cells. Thus, miRNAs might provide a basis for novel RNA anti-cancer agents.

title: Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples.
abstract: microRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 3' UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using high-throughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 mug of fresh frozen and 1-5 mug of FFPE samples (correlation coefficient R (2) = 0.86-0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.

title: Genetic alterations during colorectal-tumor development.
abstract: Because most colorectal carcinomas appear to arise from adenomas, studies of different stages of colorectal neoplasia may shed light on the genetic alterations involved in tumor progression. We looked for four genetic alterations (ras-gene mutations and allelic deletions of chromosomes 5, 17, and 18) in 172 colorectal-tumor specimens representing various stages of neoplastic development. The specimens consisted of 40 predominantly early-stage adenomas from 7 patients with familial adenomatous polyposis, 40 adenomas (19 without associated foci of carcinoma and 21 with such foci) from 33 patients without familial polyposis, and 92 carcinomas resected from 89 patients. We found that ras-gene mutations occurred in 58 percent of adenomas larger than 1 cm and in 47 percent of carcinomas. However, ras mutations were found in only 9 percent of adenomas under 1 cm in size. Sequences on chromosome 5 that are linked to the gene for familial adenomatous polyposis were not lost in adenomas from the patients with polyposis but were lost in 29 to 35 percent of adenomas and carcinomas, respectively, from other patients. A specific region of chromosome 18 was deleted frequently in carcinomas (73 percent) and in advanced adenomas (47 percent) but only occasionally in earlier-stage adenomas (11 to 13 percent). Chromosome 17p sequences were usually lost only in carcinomas (75 percent). The four molecular alterations accumulated in a fashion that paralleled the clinical progression of tumors. These results are consistent with a model of colorectal tumorigenesis in which the steps required for the development of cancer often involve the mutational activation of an oncogene coupled with the loss of several genes that normally suppress tumorigenesis.

title: Over- and under-expressed microRNAs in human colorectal cancer.
abstract: MicroRNAs (miRNAs) constitute a class of small (21-23 nucleotides) noncoding RNAs that function as post-transcriptional gene regulators. It is becoming increasingly clear that altered miRNA expression correlates with the pathogenesis of cancers. The purpose of this study was to determine the up-regulated miRNAs in human colorectal cancer. Total RNA was isolated from cancer tissues and corresponding noncancerous tissues from surgically resected colorectal cancers. The expression profiles of miRNAs were determined using a miRNA microarray containing 455 human miRNA probes. The expression status of selected miRNAs in paired clinical samples was then investigated by real-time RT-PCR. Twenty-one miRNAs were identified by miRNA array analysis as overexpressed in colorectal cancer tissues compared to normal epithelial tissues. Among them, the expression of miR-31, miR-183, miR-17-5p, miR-18a, miR-20a and miR-92 were confirmed to be significantly higher in cancer tissues than in normal tissues (P<0.05). In contrast, the expression of miR-143 and miR-145 in cancer tissues were significantly lower than in normal tissues (P<0.05). The miR-18a overexpression group tended to have a poorer clinical prognosis than the low expression group (P=0.07). We identified miRNAs that were overexpressed or under-expressed in colorectal cancers and which may be correlated with colorectal carcinogenesis.

title: miR-7 inhibits colorectal cancer cell proliferation and induces apoptosis by targeting XRCC2
abstract: Analysis using publicly available algorithms predicts that X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2), a key component in the homologous recombination repair pathway, is a potential target of micro-ribonucleic acid-7 (miR-7). Some studies have shown that both miR-7 and XRCC2 are associated with cancer development. For this purpose, we searched for the possible relationship between miR-7 and XRCC2 in the development of colorectal cancer (CRC).  miR-7 expression was assessed in CRC specimens and cell lines using real-time polymerase chain reaction (PCR). Luciferase reporter assay was used to confirm the target associations. The effect of miR-7 on cell proliferation and apoptosis was confirmed in vitro by the methylthiazol tetrazolium (MTT) assay, colony formation assay, and flow cytometry. Gene and protein expression were examined using real time PCR and western blotting, respectively. Results miR-7 was downregulated in CRC specimens and cell lines, and targeted the 3′ untranslated region of XRCC2. miR-7 overexpression reduced cyclin D1 expression and increased p21, caspase-3, and BAX expression, which subsequently inhibited CRC cell proliferation and induced CRC cell apoptosis. However, XRCC2 can repress the inhibitory effects of miR-7 on proliferation.  Our findings suggest that miR-7 plays a protective role by inhibiting proliferation and increasing apoptosis of CRC cells. It may identify new targets for anti-cancer treatment.

title: microRNA-7 is a novel inhibitor of YY1 contributing to colorectal tumorigenesis
abstract: Using microRNA (miRNA) expression array, we identified that miR-7 was deregulated in colorectal cancer (CRC). We studied the biological role and molecular target of miR-7 in CRC. miR-7 was downregulated in six out of seven colon cancer cell lines. Ectopic expression of miR-7 suppressed colon cancer cell proliferation (P<0.05), induced apoptosis (P<0.05) and caused cell-cycle arrest in G1 phase (P<0.05). The tumor suppressive function of miR-7 was further confirmed in nude mice (P<0.05). The 3′-untranslated region (3′UTR) of Yin Yang 1 (YY1) mRNA contains an evolutionarily conserved miR-7 binding site using in silico searches, luciferase reporter assay and western blot analysis confirmed that miR-7 directly bound to YY1 3′UTR to negatively regulate the protein expression of YY1 in colon cancer cell lines HCT116 and LOVO. Intriguingly, knock-down of YY1 in three colon cancer cell lines (HCT116, LOVO and DLD1) consistently suppressed cell proliferation (P<0.01) and induced apoptosis (P<0.01), indicating the opposite functions of miR-7 and YY1 in CRC. Consistent with these data, ectopic expression of YY1 promoted cell growth by increasing proliferation (P<0.01) and suppressing apoptosis (P<0.001). The tumorigenic ability of YY1 was further confirmed in vivo in xenograft-nude mouse model (P<0.01). In addition, pathway analyses revealed that the oncogenic effect by YY1 was associated with inhibiting p53 and modulating its downstream effectors p15, caspase cascades and C-Jun, and activating Wnt signaling pathway through activating β-catenin, anti-apoptotic survivin and fibroblast growth factor 4. Furthermore, multivariate analysis revealed that patients with YY1 protein high expression had a significant decrease in overall survival, and Kaplan–Meier survival curves showed that these patients had significantly shorter survival than others (P<0.0001). In conclusion, MiR-7 is a novel miRNA with tumor suppressive function in colon cancer by targeting oncogenic YY1. YY1 promotes colon cancer growth through inhibiting p53 and promoting Wnt signaling pathways and serves as an independent prognostic biomarker for CRC patients.

title: PAX6, a Novel Target of microRNA-7, Promotes Cellular Proliferation and Invasion in Human Colorectal Cancer Cells
abstract: BackgroundPaired box 6 (PAX6), a highly conserved transcriptional factor, has been implicated in tumorigenesis.AimWe aimed to explore the roles and molecular mechanisms of PAX6 and microRNA (miR-7) in colorectal cancer cells.MethodsTissue microarray immunohistochemistry and Western blot were applied to examine the PAX6 expression. Real-time RT-PCR and Western blot were performed to determine the expression of miR-7 and PAX6. Luciferase reporter assay was used to determine whether PAX6 was a target of miR-7. Effects of miR-7 and PAX6 on colorectal cell proliferation, cell cycle progression, colony formation and invasion were then investigated. Western blot was used to determine the activities of the ERK and PI3K signal pathways, as well as the protein expression of MMP2 and MMP9.ResultsThe protein levels of PAX6 were gradually increased, while the expression of miR-7 was gradually reduced with malignancy of colorectal cancer. PAX6 was further identified as a target of miR-7, and its protein expression was negatively regulated by miR-7 in human colorectal cancer cells. Overexpression of PAX6 in Caco-2 and SW480 cells enhanced cellular proliferation, cell cycle progression, colony formation, and invasion, while miR-7 upregulation repressed these biological processes. Furthermore, the activities of ERK and PI3K signal pathways, as well as the protein levels of MMP2 and MMP9, were upregulated in PAX6-overexpressed Caco-2 and SW480 cells but deregulated in miR-7-overexpressed Caco-2 and SW480 cells.ConclusionsOur study suggests that as a novel target of miR-7, PAX6 may serve as a promising therapeutic target for colorectal cancer.

title: MiR-9, -31, and -182 deregulation promote proliferation and tumor cell survival in colon cancer.
abstract: Several microRNAs (miRNAs) are known to be deregulated in colon cancer, but the mechanisms behind their potential involvement on proliferation and tumor cell survival are unclear. The present study aimed to identify miRNAs with functional implications for development of colon cancer. The cell proliferation and apoptosis were examined following perturbations of miRNA levels by employing a comprehensive miRNA library screen. miRNAs nominated for relevance to colon cancer were validated on expression and functional levels. By integrating the effect of miRNA up-regulation with the endogenous miRNA expression levels within the HT29, HCT116, and SW480 colon cancer cell lines, we identified miRNAs controlling cell proliferation (n = 53) and apoptosis (n = 93). From these functionally nominated miRNAs, we narrowed the list to 10 oncogene- and 20 tumor suppressor-like miRNAs that were also differentially expressed between colon cancer (n = 80) and normal colonic mucosa (n = 20). The differential expressions of miR-9, miR-31, and miR-182 were successfully validated in a series of colon carcinomas (n = 30) and polyps (n = 10) versus normal colonic mucosa (n = 10), whereas the functional effect was confirmed in an in-depth validation using different cell viability and apoptotic markers. Several transcription factors and genes regulating cell proliferation were identified as putative target genes by integrative miRNA/mRNA expression analysis obtained from the same colon cancer patient samples. This study suggests that deregulated expression of miR-9, miR-31, and miR-182 during carcinogenesis plays a significant role in the development of colon cancer by promoting proliferation and tumor cell survival.

title: MicroRNA-9 up-regulation is involved in colorectal cancer metastasis via promoting cell motility
abstract: Human microRNA-9 (miR-9) has been reported to be involved in the metastasis of several malignancies including brain breast cancer. However, its role in the metastasis of colorectal cancer (CRC) remains to be revealed. Here, we evaluated miR-9 expression in metastatic CRC and investigated its effects on the motility and proliferation of RKO cells. The expressions of miR-9 in 15 primary CRC specimens without distant metastasis (NM group) and 10 primary CRC specimens (M group) with distant metastasis (M group) were determined by quantitative real-time PCR. The alternations in the motility and morphology of RKO cells before and after miR-9 transfection were analyzed by migration assay and F-actin staining. The relationship between miR-9 and α-catenin was identified by Western blotting. Cell growth was examined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) assay. Significant difference of miR-9 expression was observed in M group compared to the NM group (P < 0.001). Ectopic expression of miR-9 enhanced the motility of RKO cells as well as changed their morphological appearance, while cell growth remained unchanged. The overexpression of miR-9 could also down-regulate α-catenin expression. These data suggest that miR-9 may potentially participate in the metastatic process of CRC though facilitating cell motility.

title: MicroRNA-10b is a Prognostic Indicator in Colorectal Cancer and Confers Resistance to the Chemotherapeutic Agent 5-Fluorouracil in Colorectal Cancer Cells
abstract: PurposeRecent evidence has shown that altered patterns of microRNA (miRNA) expression correlate with various human cancers. We investigated the clinical significance of miR-10b and its involvement in chemotherapeutic resistance to 5-fluorouracil (5-FU), which is a key component of common chemotherapy regimens in colorectal cancer.MethodsQuantitative RT-PCR was used to evaluate the clinicopathologic significance of miR-10b expression in 88 colorectal cancer cases. We also investigated the chemotherapeutic sensitivity to 5-FU in miR-10b-overexpressing colorectal cancer cells. To explore the mechanism of chemoresistance in miR-10b transfected cells, we examined whether miR-10b inhibits the pro-apoptotic BH3-only Bcl-2 family member BIM(BCL2L11), a key mediator of chemotherapy-induced cell death.ResultsHigh level miR-10b expression was found to be significantly associated with high incidence of lymphatic invasion (P = 0.0257) and poor prognosis (P = 0.0057). Multivariate analysis indicated that high miR-10b expression is an independent prognostic factor for survival. In vitro studies revealed that miR-10b directly inhibits pro-apoptotic BIM, and the overexpression of miR-10b confers chemoresistance in colorectal cancer cells to 5-FU.ConclusionsMiR-10b is a novel prognostic marker in colorectal cancer. Moreover, the expression of miR-10b is a potential indicator of chemosensitivity to the common 5-FU-based chemotherapy regimen.

title: microRNA-16 represses colorectal cancer cell growth in vitro by regulating the p53/survivin signaling pathway.
abstract: Dysregulated expression of microRNAs (miRNA) is a hallmark of cancer. miR-16             has been reported to be downregulated and to act as a tumor suppressor in different             cancer types. In the present study, we sought to investigate the possible roles             and mechanisms of miR-16 and its relationship with p53 and survivin in CRC cells.             We showed that miR-16 was downregulated in 67% of CRC tissues and was correlated             with the degree of histological differentiation. Experiments in vitro showed that             overexpression of miR-16 inhibited the proliferation and induced apoptosis of             CRC cells through the intrinsic apoptosis pathway. We further showed that miR-16             repressed survivin expression at both the mRNA and protein levels and the survivin             gene was a direct target of miR-16. In addition, miR-16 reduced p53 expression             and p53 increased miR-16 levels, with downregulation of miR-16 targets survivin,             cyclin D1 and CDK6. Our findings suggest that miR-16 represses colorectal cancer             cell growth in vitro by regulating the p53/survivin signaling pathway. Our findings             provide further evidence for the involvement of dysregulated miRNAs in CRC, and             miR-16 could serve as a molecular target for CRC therapy.

title: A microRNA expression signature of human solid tumors defines cancer gene targets
abstract: Small noncoding microRNAs (miRNAs) can contribute to cancer development and progression and are differentially expressed in normal tissues and cancers. From a large-scale miRnome analysis on 540 samples including lung, breast, stomach, prostate, colon, and pancreatic tumors, we identified a solid cancer miRNA signature composed by a large portion of overexpressed miRNAs. Among these miRNAs are some with well characterized cancer association, such as miR-17-5p, miR-20a, miR-21, miR-92, miR-106a, and miR-155. The predicted targets for the differentially expressed miRNAs are significantly enriched for protein-coding tumor suppressors and oncogenes (P < 0.0001). A number of the predicted targets, including the tumor suppressors RB1 (Retinoblastoma 1) and TGFBR2 (transforming growth factor, beta receptor II) genes were confirmed experimentally. Our results indicate that miRNAs are extensively involved in cancer pathogenesis of solid tumors and support their function as either dominant or recessive cancer genes.

title: Multiple microRNAs induced by Cdx1 suppress Cdx2 in human colorectal tumour cells.
abstract: The mammalian transcriptional factors, Cdx1 and Cdx2 (Cdx is caudal-type homeobox) are paralogues and critical for the cellular differentiation of intestinal or colorectal epithelia. It has been reported previously that in Cdx1 transgenic or knockout mice, endogenous Cdx2 levels are inversely correlated with Cdx1 levels. Recently, we found that exogenous Cdx1 expression can suppress Cdx2 in a human colorectal tumour cell line, SW480, although the underlying molecular mechanisms were unclear. In the present study, we show that several microRNAs induced by exogenous Cdx1 expression directly bind to the CDX2 mRNA 3'UTR (untranslated region) to destabilize these transcripts, finally leading to their degradation. Using microarray analysis, we found that several miRNAs that were computationally predicted to target CDX2 mRNAs are up-regulated by exogenous Cdx1 expression in SW480 cells. Among these molecules, we identified miR-9, miR-16 and miR-22 as having the potential to suppress Cdx2 through the binding of the 3'UTR to its transcript. Importantly, simultaneous mutations of both the miR-9- and miR-16-binding sites in the CDX2 3'UTR were shown to be sufficient to block Cdx2 suppression. The results of the present study suggest a unique feature of miRNAs in which they contribute to homoeostasis by limiting the levels of transcription factors belonging to the same gene family.

title: mRNA/microRNA gene expression profile in microsatellite unstable colorectal cancer
abstract: BackgroundColorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome.ResultsWe investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H) for genome-wide expression of microRNA (miRNA) and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response.ConclusionThis is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

title: Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer
abstract: BackgroundMicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis.ResultsThe expression analysis of clinical CRC samples identified 37 miRNAs that were differentially expressed between CRC and normal tissue. Furthermore, several of these miRNAs were associated with CRC tumor progression including loss of miR-133a and gain of miR-224. We identified 11 common miRNAs that were differentially expressed between normal colon and CRC in both the cell line models and clinical samples. In vitro functional studies indicated that miR-143 and miR-145 appear to function in opposing manners to either inhibit or augment cell proliferation in a metastatic CRC model. The pathways targeted by miR-143 and miR-145 showed no significant overlap. Furthermore, gene expression analysis of metastatic versus non-metastatic isogenic cell lines indicated that miR-145 targets involved in cell cycle and neuregulin pathways were significantly down-regulated in the metastatic context.ConclusionMiRNAs showing altered expression at different stages of CRC could be targets for CRC therapies and be further developed as potential diagnostic and prognostic analytes. The identified biological processes and signalling pathways collectively targeted by co-expressed miRNAs in CRC provide a basis for understanding the functional role of miRNAs in cancer.

title: MiR-17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression
abstract: MicroRNAs are small non-coding RNA molecules, which regulate central mechanisms of tumorigenesis. In colorectal tumours, the combination of gain of 8q and 13q is one of the major factors associated with colorectal adenoma to adenocarcinoma progression. Functional studies on the miR-17-92 cluster localised on 13q31 have shown that its transcription is activated by c-myc, located on 8q, and that it has oncogenic activities. We investigated the contribution of the miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression.Expression levels of the miR-17-92 cluster were determined in 55 colorectal tumours and in 10 controls by real-time RT–PCR. Messenger RNA c-myc expression was also determined by real-time RT–PCR in 48 tumours with array comparative genomic hybridisation (aCGH) data available.From the six members of the miR-17-92 cluster, all except miR-18a, showed significant increased expression in colorectal tumours with miR-17-92 locus gain compared with tumours without miR-17-92 locus gain. Unsupervised cluster analysis clustered the tumours based on the presence of miR-17-92 locus gain. Significant correlation between the expression of c-myc and the six miRNAs was also found.Increased expression of miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression is associated to DNA copy number gain of miR17-92 locus on 13q31 and c-myc expression.

title: The colorectal microRNAome.
abstract: MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.

title: Human colon cancer profiles show differential microRNA expression depending on mismatch repair status and are characteristic of undifferentiated proliferative states
abstract: BackgroundColon cancer arises from the accumulation of multiple genetic and epigenetic alterations to normal colonic tissue. microRNAs (miRNAs) are small, non-coding regulatory RNAs that post-transcriptionally regulate gene expression. Differential miRNA expression in cancer versus normal tissue is a common event and may be pivotal for tumor onset and progression.MethodsTo identify miRNAs that are differentially expressed in tumors and tumor subtypes, we carried out highly sensitive expression profiling of 735 miRNAs on samples obtained from a statistically powerful set of tumors (n = 80) and normal colon tissue (n = 28) and validated a subset of this data by qRT-PCR.ResultsTumor specimens showed highly significant and large fold change differential expression of the levels of 39 miRNAs including miR-135b, miR-96, miR-182, miR-183, miR-1, and miR-133a, relative to normal colon tissue. Significant differences were also seen in 6 miRNAs including miR-31 and miR-592, in the direct comparison of tumors that were deficient or proficient for mismatch repair. Examination of the genomic regions containing differentially expressed miRNAs revealed that they were also differentially methylated in colon cancer at a far greater rate than would be expected by chance. A network of interactions between these miRNAs and genes associated with colon cancer provided evidence for the role of these miRNAs as oncogenes by attenuation of tumor suppressor genes.ConclusionColon tumors show differential expression of miRNAs depending on mismatch repair status. miRNA expression in colon tumors has an epigenetic component and altered expression that may reflect a reversion to regulatory programs characteristic of undifferentiated proliferative developmental states.

title: Initial study of microRNA expression profiles of colonic cancer without lymph node metastasis
abstract: To investigate the difference of microRNA expression profiles between colonic cancer without lymph node metastasis and the para‐cancerous control, to identify the specific microRNA associated with the cancer and to predict the carcinogenetic mechanism of microRNA on the basis of these results.

title: MicroRNA-18a Attenuates DNA Damage Repair through Suppressing the Expression of Ataxia Telangiectasia Mutated in Colorectal Cancer
abstract: miR-18a is one of the most up-regulated miRNAs in colorectal cancers (CRC) based on miRNA profiling. In this study, we examined the functional significance of miR-18a in CRC.  Expression of miR-18a was investigated in 45 CRC patients. Potential target genes of miR-18a were predicted by in silico search and confirmed by luciferase activity assay and Western blot. DNA damage was measured by comet assay. Gene function was measured by cell viability, colony formation and apoptosis assays.  The up-regulation of miR-18a was validated and confirmed in 45 primary CRC tumors compared with adjacent normal tissues (p<0.0001). Through in silico search, the 3′UTR of Ataxia telangiectasia mutated (ATM) contains a conserved miR-18a binding site. Expression of ATM was down-regulated in CRC tumors (p<0.0001) and inversely correlated with miR-18a expression (r = -0.4562, p<0.01). Over-expression of miR-18a in colon cancer cells significantly reduced the luciferase activity of the construct with wild-type ATM 3′UTR but not that with mutant ATM 3′UTR, inferring a direct interaction of miR-18a with ATM 3′UTR. This was further confirmed by the down-regulation of ATM protein by miR-18a. As ATM is a key enzyme in DNA damage repair, we evaluated the effect of miR-18a on DNA double-strand breaks. Ectopic expression of miR-18a significantly inhibited the repair of DNA damage induced by etoposide (p<0.001), leading to accumulation of DNA damage, increase in cell apoptosis and poor clonogenic survival.  miR-18a attenuates cellular repair of DNA double-strand breaks by directly suppressing ATM, a key enzyme in DNA damage repair.

title: MicroRNA-19a targets tissue factor to inhibit colon cancer cells migration and invasion
abstract: The over-expression of tissue factor (TF) and its roles in colon cancer progression have attracted much attention. However, the mechanisms regulating TF expression have not yet been shown in detail. In this study, we over-expressed miR-19a, miR20a and miR-106b in colon cancer cells, and evaluated their impact on TF expression and cellular function. We provide evidence demonstrating that miR-19a inhibited TF expression in vitro. Luciferase reporter assay confirmed that TF was a direct target of miR-19a because the miR-19a mediated repression of luciferase activity was abolished by mutation of the putative binding site. Moreover, miR-19a suppressed colon cancer cell migration and invasion. This effect was due to the indirect down-regulation of matrix metalloproteinase 9. Finally, we investigated the relevance of TF and miR-19a expression in a total of 48 paired colon cancer samples and revealed that miR-19a was inversely correlated with TF expression in stages I and II cases. Therefore, our results suggested that miR-19a was capable of suppressing TF expression in vitro and inhibiting cell migration and invasion. Although it was not the unique mechanism responsible for the expression of TF in vivo, miR-19a was inversely correlated with TF expression in early stage colon cancer patients.

title: Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells
abstract: Drug resistance in colorectal cancers is assumed to be mediated by changes in the expression of microRNAs, but the specific identities and roles of microRNAs are largely unclear. We examined the effect of 5-fluorouracil (5-FU) resistance on microRNA expression.  Two types of 5-FU-resistant colon cancer cells were derived from the DLD-1 and KM12C cell lines. The expressions of microRNAs were profiled with a microarray containing 723 microRNAs and validated by quantitative real-time polymerase chain reaction (qRT-PCR). To survey the downstream mediators of microRNA, we used a microRNA:mRNA immunoprecipitation (RIP)-Chip and pathway analysis tool to identify potential direct targets of microRNA.  In response to 5-FU, miR-19b and miR-21 were over-expressed in 5-FU-resistant cells. Of note, miR-19b was up-regulated 3.47-fold in the DLD-1 resistant cells, which exhibited no alteration in cell cycle profiles despite exposure to 5-FU. After transfection of miR-19b, specific mRNAs were recruited to microRNA:mRNA complexes isolated with Ago2 antibody and subjected to whole-genome transcriptional analysis. In this analysis, 66 target mRNAs were enriched by at least 5.0-fold in the microRNA:mRNA complexes from DLD-1 resistant cells. Ingenuity pathway analysis of mRNA targets significantly (P \ 0.05) indicated the category ‘‘Cell Cycle’’ as a probable area of the molecular and cellular function related with 5-FU resistance. Among candidate mRNA targets, SFPQ and MYBL2 have been linked to cell cycle functions.  We revealed up-regulation of miR-19b in response to 5-FU and potential targets of miR-19b mediating the cell cycle under treatment with 5-FU. Our study provides an important insight into the mechanism of 5-FU resistance in colorectal cancers.

title: Screening of differentially expressed microRNA in ulcerative colitis related colorectal cancer.
abstract: To investigate the differential expression of microRNA (miRNA) in colon between ulcerative colitis (UC) and ulcerative colitis related colorectal cancer (UCRCC).



An UC mouse model was built by dextran sodium sulfate, and an UCRCC mouse model by dextran sodium sulfate and 1,2-diformylhydrazine. RNAs were extracted from the colon, purified and hybridized with fluorescence-labeled miRNA oligonucleotide gene chip. Real-time fluorescence quantitative PCR was used to verify the expression variation of miRNA. SAM was employed for the data analysis.



The up-regulated miRNAs in colon cancer included has-miR-194, has-miR-215, has-miR-93, has-miR-192, has-miR-92a, has-miR-29b, and has-miR-20a (median false discovery rate<5%), while the down-regulated miRNAs were has-miR-1231, has-miR-195, has-miR-143, and has-miR-145 (median false discovery rate<5%).



Significant differential expression of miRNA was found between the UC mouse and UCRCC mouse, which may be related to the onset, erosion and transfer of colorectal cancer.

title: The Prognostic Value of MicroRNAs Varies with Patient Race/Ethnicity and Stage of Colorectal Cancer
abstract: MicroRNAs (miRNA) have potential prognostic value for colorectal cancers; however, their value based on patient race/ethnicity and pathologic stage has not been determined. The goal was to ascertain the prognostic value of 5 miRNAs with increased expression in colorectal cancers of African American (black) and non-Hispanic Caucasian (white) patients.  TaqMan quantitative real-time PCR was used to quantify expression of miR-20a, miR-21, miR-106a, miR-181b, and miR-203 in paired normal and tumor colorectal cancer archival tissues collected from 106 black and 239 white patients. The results were correlated with overall survival based on patient race/ethnicity and pathologic stage. Because decisions about adjuvant therapy are important for stage III colorectal cancers, and because miR-181b seemed to have prognostic value only for stage III black patients, we assessed its prognostic value in a separate cohort of 36 stage III colorectal cancers of blacks.  All 5 miRNAs had higher expression in colorectal cancers (>1.0-fold) than in corresponding normal tissues. High expression of miR-203 was associated with poor survival of whites with stage IV colorectal cancers (HR = 3.00; 95% CI, 1.29–7.53), but in blacks it was an indicator of poor survival of patients with stages I and II colorectal cancers (HR = 5.63; 95% CI, 1.03–30.64). Increased miR-21 expression correlated with poor prognosis for white stage IV patients (HR = 2.50; 95% CI, 1.07–5.83). In both test and validation cohorts, high miR-181b expression correlated with poor survival of only black patients with stage III colorectal cancers (HR = 1.94; 95% CI, 1.03–3.67).  These preliminary findings suggest that the prognostic value of miRNAs in colorectal cancers varies with patient race/ethnicity and stage of disease.

title: Clinical, Pathologic, and Molecular Features of Early-onset Colorectal Carcinoma
abstract: The incidence of colorectal carcinoma has increased among patients <40 years of age for unclear reasons. In this study, we describe the clinical, pathologic, and molecular features of colorectal carcinomas that developed in young patients. We compiled a study group of 24 patients <40 years of age with colorectal carcinoma, and 45 patients ≥40 years of age served as controls. Cases were evaluated for clinical risk factors of malignancy and pathologic features predictive of outcome. The tumors were immunohistochemically stained for O6-methylguanine methyltransferase, MLH-1, MSH-2, MSH-6, β-catenin, chemokine (C-X-C motif) receptor 4, epidermal growth factor receptor, TP53, p16, survivin, and α-methylacyl-CoA racemase; assessed for microsatellite instability and mutations in β-catenin, APC, EGFR, PIK3CA, KRAS, and BRAF; evaluated for micro-RNA expression (miR-21, miR-20a, miR-183, miR-192, miR-145, miR-106a, miR-181b, and miR-203); and examined for evidence of human papillomavirus infection. One study patient each had ulcerative colitis and hereditary nonpolyposis colorectal cancer. Ninety-two percent of tumors from young patients occurred in the distal colon (P=0.006), particularly the rectum (58%, P=0.02), and 75% were stage III or IV. Tumors from young patients showed more frequent lymphovascular (81%, P=0.03) and/or venous (48%, P=0.003) invasion, an infiltrative growth pattern (81%, P=0.03), and α-methylacyl-CoA racemase expression (83%, P=0.02) compared with controls. Carcinomas in this group showed significantly increased expression of miR-21, miR-20a, miR-145, miR-181b, and miR-203 (P≤0.005 for all comparisons with controls). These results indicate that early-onset carcinomas commonly show pathologic features associated with aggressive behavior. Posttranslational regulation of mRNA and subsequent protein expression may be particularly important to the development of colorectal carcinomas in young patients.

title: Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro.
abstract: MicroRNAs (miRNAs) are small noncoding RNA molecules produced by miR genes which are able to control the expression of a large number of cellular proteins by targeting mRNAs of protein coding genes. It has been suggested that modification of miR gene expression could be an important factor in the development and maintenance of the neoplastic state. It is also reasonable to hypothesize that antineoplastic drugs could be able to alter miR gene expression pattern since most of them are able to interfere with nucleic acid metabolism and gene expression. Here we show that 5-fluorouracil (5-FU), a classical antimetabolite largely used in the clinic, is able to change significantly the expression of several miR genes. In colon cancer cells, at a clinically relevant concentration, the drug up-regulates or down-regulates in vitro the expression of 19 and 3 miR genes, respectively, by a factor of not less than two-fold. In some instances, 5-FU up-regulates miR genes that are already over-expressed in neoplastic tissues, including, for example, miR-21 that is associated with anti-apoptotic functions characterizing malignant cells. In this case, it is possible that drug-induced miR gene dysregulation could be the expression of cellular response to the toxic effects of the agent. On the contrary, in other instances the drug influences the expression of miR genes in a direction that is opposite to that induced by neoplastic transformation. A typical example is provided by miR-200b, that is up-regulated in various tumors and down-regulated by treatment with the antimetabolite. Noteworthy, it is known that miR-200b suppresses a gene that codes for a protein tyrosine phosphatase (PTPN12) that inactivates products of oncogenes, such as c-Abl, Src or Ras. In conclusion, the present results support the hypothesis that 5-FU can alter profoundly miR gene expression pattern. This effect could be responsible, at least in part, of the multi-target pleiotropic influence manifested by the drug on malignant cells.

title: miR-185 and miR-133b deregulation is associated with overall survival and metastasis in colorectal cancer.
abstract: Colorectal cancer (CRC) is one of the most common and deadly forms of cancer. Despite improved treatment modalities, post-operative recurrence and metastasis remain the major problems for extending patient survival after surgery. This highlights the need to search for biomarkers for prognostication and treatment stratification of colorectal cancer patients. In this study, we applied the SYBR-green quantitative PCR-based array approach to screen for differentially expressed miRNAs between patients with short (<50 months, range 10-33 months) and long survival (≥ 50 months, range 50-152 months). The selected candidate prognostic miRNAs were validated in a cohort of 50 CRC patients by TaqMan quantitative PCR. We found that high expression of miR-185 and low expression of miR-133b were correlated with poor survival (p=0.001 and 0.028, respectively) and metastasis (p=0.007 and 0.036, respectively) in colorectal cancer. Our findings suggest the potential prognostic values of these miRNAs for predicting clinical outcome after surgery.

title: miR-185 targets RhoA and Cdc42 expression and inhibits the proliferation potential of human colorectal cells.
abstract: Increasing evidence in the past few years has shown that miRNAs could serve functionally as "oncogenes" or "tumor suppressor genes" and regulate multiple cellular processes relevant to carcinogenesis and cancer progression. Both RhoA and Cdc42, two members of the Rho GTPase family, are found to be upregulated in several types of human tumors including colorectal cancer, and have been implicated in cancer initiation and progression. In the present studies, we found that miR-185 expression greatly inhibited the proliferation potential of Hela cells. An examination of the predicted targets of miR-185 revealed RhoA and Cdc42 among the putative targets that are crucial for cell proliferation. A genomic sequence analysis indicated that nt 1844-1852 of the RhoA 3'UTR and nt 1382-1396 of the cdc42 3'UTR encode for miR-185 target matching sequences and they are highly conserved across different species. Using a luciferase-reporter assay, we show that miR-185 expression significantly suppressed the RhoA and Cdc42 3'UTR activities, and the inhibitory effect was lost when the putative target sites for miR-185 were mutated. Consistent with these results, ectopic expression of miR-185 reduced protein levels of RhoA and Cdc42 in cells, indicating miR-185 functionally regulates RhoA and Cdc42 abundance. Similar to the effects of knocking down RhoA and/or Cdc42 expression, miR-185 effectively inhibited proliferation, induced G1 cell cycle arrest and apoptosis, and blocked invasion of colorectal cancer cells. Thus, miR-185 is a negative regulator of RhoA and Cdc42 and their cellular activities, and could inhibit proliferation and invasion of colorectal cancer cells.

title: p53-Responsive micrornas 192 and 215 are capable of inducing cell cycle arrest.
abstract: microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.

title: miR-192 Regulates Dihydrofolate Reductase and Cellular Proliferation through the p53-microRNA Circuit
abstract: The purpose of this study is to investigate the molecular mechanism of miR-192 in colon cancer.  Human colon cancer cell lines with different p53 status were used as our model system to study the effect of miR-192 on cell proliferation, cell cycle control, and mechanism of regulation.  Our results show that one of the key miR-192 target genes is dihydrofolate reductase (DHFR). miR-192 affects cellular proliferation through the p53-miRNA circuit. Western immunoblot analyses indicated that the expression of DHFR was significantly decreased by miR-192. Further investigation revealed that such suppression was due to translational arrest rather than mRNA degradation. More profound inhibition of cellular proliferation was observed by ectopic expression of miR-192 in colon cancer cell lines containing wild-type p53 than cells containing mutant p53. Thus, the effect of miR-192 on cellular proliferation is mainly p53 dependent. Overexpression of miR-192 triggered both G1 and G2 arrest in HCT-116 (wt-p53) cells but not in HCT-116 (null-p53) cells. The cell cycle checkpoint control genes p53 and p21 were highly overexpressed in cells that overexpressed miR-192. Endogenous miR-192 expression was increased in HCT-116 (wt-p53) and RKO (wt-p53) cells treated with methotrexate, which caused an induction of p53 expression. Chromatin immunoprecipitation-quantitative reverse transcription-PCR analysis revealed that the p53 protein interacted with the miR-192 promoter sequence.  These results indicate that miR-192 may be another miRNA candidate that is involved in the p53 tumor suppressor network with significant effect on cell cycle control and cell proliferation.

title: MicroRNA-192 Suppresses Liver Metastasis of Colon Cancer
abstract: Metastasis causes most deaths from colon cancer yet mechanistic understanding and therapeutic options remain limited. Here we show that expression of microRNA (miR)-192 is inversely correlated with metastatic potential of colon cancer cells. Ectopic expression of miR-192 sensitizes colon cancer cells to growth factor deprivation stress-induced apoptosis, whereas inhibition of miR-192 confers resistance. Overexpression of miR-192 inhibits metastatic colonization to the liver in an orthotopic mouse model of colon cancer. Alterations associated with the metastatic phenotype in the primary tumors include increased apoptosis, decreased proliferation and angiogenesis. Further studies indicate that miR-192 downregulates expression of Bcl-2, Zeb2 and VEGFA in vitro and in vivo, which is responsible for enhanced apoptosis, increased expression of E-cadherin and decreased angiogenesis in vivo, respectively. Finally, studies performed on human colonic adenocarcinoma show that expression of miR-192 is significantly reduced in neoplastic cells as compared with normal colonic epithelium. Importantly, there is a significant decrease in miR-192 expression in stage IV tumors when compared with stage I or II lesions. These findings indicate that miR-192 has an important role in colon cancer development and progression. Our studies underscore the clinical relevance and prognostic significance of miR-192 expression in colon cancer. Therefore, a major implication of our studies is that restoration of miR-192 expression or antagonism of its target genes (Bcl-2, Zeb2 or VEGFA) may have considerable therapeutic potential for anti-metastatic therapy in patients with colon cancer.

title: microRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells.
abstract: Deregulated microRNAs and their roles in cancer development have attracted much attention. In the present study, we analyzed the roles of miR-195 in colorectal cancer pathogenesis, as its participation in some other types of cancer has been suggested by previous reports. By comparing miR-195 expression in 81 human colorectal cancer tissues and matched non-neoplastic mucosa tissues, we found that miR-195 was downregulated in cancer tissues. And restoration of miR-195 in colorectal cancer cell lines HT29 and LoVo could reduce cell viability, promote cell apoptosis and suppress tumorigenicity. Moreover, important antiapoptotic Bcl-2 was identified to be directly targeted by miR-195, and miR-195 was further suggested to exert its proapoptotic function mainly through targeting Bcl-2 expression. Taken together, our study provides important roles of miR-195 in colorectal cancer pathogenesis and implicates its potential application in cancer therapy.

title: High miR-196a levels promote the oncogenic phenotype of colorectal cancer cells.
abstract: To analyze the relevance of the microRNA miR-196a for colorectal oncogenesis.



The impact of miR-196a on the restriction targets HoxA7, HoxB8, HoxC8 and HoxD8 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) after transient transfection of SW480 cancer cells. The miR-196a transcription profile in colorectal cancer samples, mucosa samples and diverse cancer cell lines was quantified by RT-PCR. Transiently miR-196a-transfected colorectal cancer cells were used for diverse functional assays in vitro and for a xenograft lung metastasis model in vivo.



HoxA7, HoxB8, HoxC8 and HoxD8 were restricted by miR-196a in a dose-dependent and gene-specific manner. High levels of miR-196a activated the AKT signaling pathway as indicated by increased phosphorylation of AKT. In addition, high levels of miR-196a promoted cancer cell detachment, migration, invasion and chemosensitivity towards platin derivatives but did not impact on proliferation or apoptosis. Furthermore, miR-196a increased the development of lung metastases in mice after tail vein injection.



miR-196a exerts a pro-oncogenic influence in colorectal cancer.

title: Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer
abstract: Axl is a receptor that induces proliferation, migration and invasion in cancer. In this study, we show that specific microRNAs (miRNAs) target the 3′-UTR of Axl. Luciferase-reporter assays with wild-type and deleted miR-34 and miR-199a/b seed sequences of Axl 3′-UTR confirmed the specificity of targeting. An inverse correlation between Axl protein and miR-34a expression in a panel of non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and breast cancer (BRC) cell lines was observed, while miR-199a/b expression was completely suppressed. Pre-miR transfection inhibited in vitro migration and invasion and, in vivo, reduced the number of distant lung- or liver-metastases in a chorion-allantoic-membrane (CAM) assay. Moreover, methylation-specific PCR on bisulfite-converted DNA obtained from the cell lines showed that the miR-34a promoter methylation status was inversely correlated with its expression, and that miR-199a/b promoter regions were methylated in all cells tested. In a panel of NSCLC tissues (n=44), miR-34a and miR-199a/b were found to be downregulated and significantly co-expressed. A lower expression of all three miRs was significantly associated with squamous histotypes, and, in a preliminary series, NSCLC patients with miR-34a upregulation showed a positive association towards a longer survival. These results indicate that Axl receptor expression can be regulated by miR-34a and miR-199a/b, which are suppressed by promoter methylation in solid cancer cells.

title: Regulation of Colorectal Carcinoma Stemness, Growth, and Metastasis by an miR-200c-Sox2–Negative Feedback Loop Mechanism
abstract: To elucidate a novel mechanism of miR-200c in the regulation of stemness, growth, and metastasis in colorectal carcinoma (CRC).  Quantitative reverse transcription PCR was used to quantify miR-200c expression in CRC cell lines and tissues. A luciferase assay was adopted for the target evaluation. The functional effects of miR-200c in CRC cells were assessed by its forced or inhibited expression using lentiviruses.  MiR-200c was statistically lower in CRC clinical specimens and highly metastatic CRC cell lines compared with their counterparts. Sox2 was validated as a target for miR-200c. The knockdown of miR-200c significantly enhanced proliferation, migration, and invasion in CRC cell lines, whereas the upregulation of miR-200c exhibited an inverse effect. Moreover, rescue of Sox2 expression could abolish the effect of the upregulation of miR-200c. In addition, the reduction of miR-200c increased the expression of CRC stem cell markers and the sphere-forming capacity of CRC cell lines. Further study has shown that miR-200c and Sox2 reciprocally control their expression through a feedback loop. MiR-200c suppresses the expression of Sox2 to block the activity of the phosphoinositide 3-kinase (PI3K)–AKT pathway.  Our findings indicate that miR-200c regulates Sox2 expression through a feedback loop and is associated with CRC stemness, growth, and metastasis.

title: Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases
abstract: Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.

title: The roles of miR-200c in colon cancer and associated molecular mechanisms
abstract: The expression of miR-200c has been widely reported to be elevated in tumor tissues and sera of patients with colorectal cancer (CRC) and has been found to correlate with poor prognosis. However, how miR-200c regulates the apoptosis, survival, invasion, metastasis, and tumor growth in colon cancer cells remains to be fully elucidated. This study seeks to further investigate the role of miR-200c in colon cancer development. The expression of miR-200c in tumor and peritumoral tissues of 101 colon cancer patients was measured by real-time PCR. miR-200c expression in HCT-116 and HT-29 colon cancer cells was silenced by adenovirus-carried expression of antisense mRNA against miR-200c. The protein levels of PTEN, p53 Ser15, PP1, and activated caspase-3 in HCT-116 and HT-29 cells were measured by Western blot. This study demonstrated that the expression of miR-200c was significantly higher in tumor tissues than in peritumoral tissues of colon cancer patients. The elevated miR-200c expression significantly correlated with the TNM stage, lymph node metastasis, and invasion of colon cancer. Silencing miR-200c expression significantly induced cell apoptosis, inhibited long-term survival, invasion, and metastasis, and delayed xenograft tumor growth. Importantly, silencing miR-200c expression sensitized the therapeutic effect of Ara-C (Cytarabine). The effects of silencing miR-200c expression were associated with upregulation of PTEN protein and p53 Ser15 phosphorylation levels in HCT-116 cells and PTEN protein expression in HT-29 cells. In conclusion, miR-200c functions as an oncogene in colon cancer cells through regulating tumor cell apoptosis, survival, invasion, and metastasis as well as xenograft tumor growth through inhibition of PTEN expression and p53 phosphorylation.

title: MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis
abstract: Distant metastasis is the major cause of cancer-related death in patients with colorectal cancer (CRC). Although the microRNA-200 (miR-200) family is a crucial inhibitor of epithelial-to-mesenchymal transition (EMT) in human cancer, the role of miR-200 members in the pathogenesis of metastatic CRC has not been investigated.  Fifty-four pairs of primary CRC and corresponding matched liver metastasis tissue specimens were analysed for expression and methylation status of the miR-200 family members. Functional analysis of miR-200c overexpression was investigated in CRC cell lines, and cells were analysed for proliferation, invasion and migration. Expression of several miR-200c target genes (ZEB1, ETS1 and FLT1) and EMT markers (E-cadherin and vimentin) in CRC cell lines and tissue specimens was validated.  Liver metastasis tissues showed higher expression of miR-200c (primary CRC=1.31 vs. liver metastasis=1.59; p=0.0014) and miR-141 (primary CRC=0.14 vs. liver metastasis=0.17; p=0.0234) than did primary CRCs, which was significantly associated with hypomethylation of the promoter region of these miRNAs (primary CRC=61.2% vs. liver metastasis=46.7%; p<0.0001). The invasive front in primary CRC tissues revealed low miR-200c expression by in situ hybridization analysis. Transfection of miR-200c precursors resulted in enhanced cell proliferation but reduced invasion and migration behaviours in CRC cell lines. Overexpression of miR-200c in CRC cell lines caused reduced expression of putative gene targets, and resulted in increased E-cadherin and reduced vimentin expression. The associations between miR-200c, target genes and EMT markers were validated in primary CRCs and matching liver metastasis tissues. Conclusions miR-200c plays an important role in mediating EMT and metastatic behaviour in the colon. Its expression is epigenetically regulated, and miR-200c may serve as a potential diagnostic marker and therapeutic target for patients with CRC.

title: Aberrant Expression of miR-203 and Its Clinical Significance in Gastric and Colorectal Cancers
abstract: BackgroundMicroRNAs (miRNAs) are small, non-coding RNAs of endogenous origin, they have been increasingly shown to have aberrant expression in many tumor types. miR-203 has not been comprehensively investigated in gastric and colorectal cancers.MethodsTotal RNA was extracted from tissues of 212 patients with gastric or colorectal cancer as well as from seven gastric and colorectal cell lines. We determined the expression of miR-203 by real-time PCR and calculated using the $$ {{2}^{ - }}^{{\Delta \Delta Ct}} $$ method. Then, we assessed miR-203 expression and clinicopathologic characteristics. Finally, we studied the effect of miR-203 on cell proliferation in SGC-7901 cells by MTT.ResultsmiR-203 has significantly low expression in colorectal cancer tissues (p < 0.001, paired t test) and cancer cell lines compared to non-tumor counterparts. Moreover, low expression of miR-203 was correlated with tumor size (p = 0.015, non-parametric test) and pT stage (p = 0.005) in colorectal cancer. Although expression of miR-203 was not significant in gastric cancer tissues (p = 0.124), interestingly, miR-203 was correlated with tumor size (p = 0.023), macroscopic type (p = 0.045), and pT stage (p = 0.013). Furthermore, we found miR-203 can inhibit the cell proliferation in SGC-7901 cells.ConclusionmiR-203 may be related to the proliferation and invasion of gastric and colorectal cancers.

title: miR‐203 induces oxaliplatin resistance in colorectal cancer cells by negatively regulating ATM kinase
abstract: Chemotherapy for patients with metastatic colorectal cancer (CRC) is the standard of care, but ultimately nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may help identify novel targets and drugs that will, in turn, improve therapy. Oxaliplatin is a common component combination therapeutic regimen for use in patients with metastatic CRC, but is also used as a component of adjuvant therapy for patients at risk for recurrent disease. In this study, unbiased microRNA array screening revealed that the miR‐203 microRNA is up‐regulated in three of three oxaliplatin‐resistant CRC cell lines, and therefore we investigated the role of miR‐203 in chemoresistance. Exogenous expression of miR‐203 in chemo‐naïve CRC cells induced oxaliplatin resistance. Knockdown of miR‐203 sensitized chemoresistant CRC cells to oxaliplatin. In silico analysis identified ataxia telangiectasia mutated (ATM), a primary mediator of the DNA damage response, as a potential target of miR‐203. ATM mRNA and protein levels were significantly down‐regulated in CRC cells with acquired resistance to oxaliplatin. Using TCGA database, we identified a significant reverse correlation of miR‐203 and ATM expression in CRC tissues. We validated ATM as a bona fide target of miR‐203 in CRC cells. Mutation of the putative miR‐203 binding site in the 3′ untranslated region (3′UTR) of the ATM mRNA abolished the inhibitory effect of miR‐203 on ATM. Furthermore, stable knockdown of ATM induced resistance to oxaliplatin in chemo‐naïve CRC cells. This is the first report of oxaliplatin resistance in CRC cells induced by miR‐203‐mediated suppression of ATM.

title: miR-203 reverses chemoresistance in p53-mutated colon cancer cells through downregulation of Akt2 expression.
abstract: In this study, we explored miR-203's role in the chemoresistance of colon cancer. We found that overexpression of miR-203 significantly decreased cell proliferation and survival, and induced cell apoptosis in the p53-mutated colon cancer cells. Importantly, miR-203 overexpression increased the cytotoxic role of paclitaxel in the p53-mutated colon cancer cells, but not in the p53 wild-type cells. We further demonstrated that the tumor suppressive role of miR-203 was mediated by negatively regulating Akt2 protein expression through mRNA degradation. The inhibition of Akt2 activity downregulated the protein expression of its downstream molecules involved in chemoresistance, such as MTDH and HSP90 genes. Also, overexpression of miR-203 decreased anti-apoptotic gene Bcl-xL expression and increased apoptotic proteins Bax and active caspase-3 levels. Our study is the first to identify the tumor suppressive role of overexpressed miR-203, describe its associated signaling pathways, and highlight the role of miR-203 in chemoresistance.

title: Identification and Functional Analysis of Epigenetically Silenced MicroRNAs in Colorectal Cancer Cells
abstract: Abnormal microRNA (miRNA) expression has been linked to the development and progression of several human cancers, and such dysregulation can result from aberrant DNA methylation. While a small number of miRNAs is known to be regulated by DNA methylation, we postulated that such epigenetic regulation is more prevalent. By combining MBD-isolated Genome Sequencing (MiGS) to evaluate genome-wide DNA methylation patterns and microarray analysis to determine miRNA expression levels, we systematically searched for candidate miRNAs regulated by DNA methylation in colorectal cancer cell lines. We found 64 miRNAs to be robustly methylated in HCT116 cells; eighteen of them were located in imprinting regions or already reported to be regulated by DNA methylation. For the remaining 46 miRNAs, expression levels of 18 were consistent with their DNA methylation status. Finally, 8 miRNAs were up-regulated by 5-aza-2′-deoxycytidine treatment and identified to be novel miRNAs regulated by DNA methylation. Moreover, we demonstrated the functional relevance of these epigenetically silenced miRNAs by ectopically expressing select candidates, which resulted in inhibition of growth and migration of cancer cells. In addition to reporting these findings, our study also provides a reliable, systematic strategy to identify DNA methylation-regulated miRNAs by combining DNA methylation profiles and expression data.

title: Altered Expression of miR-21, miR-31, miR-143 and miR-145 Is Related to Clinicopathologic Features of Colorectal Cancer
abstract: Development and metastases of colorectal cancer (CRC) are characterized by multiple genetic alterations. MicroRNAs (miRNAs) are endogenously expressed regulatory noncoding RNAs. Previous, mainly preclinical studies showed altered expression levels of several miRNAs in CRC.  In our study, the expression levels of miR-21, miR-31, miR-143 and miR-145 in 29 primary colorectal carcinomas and 6 non-tumor adjacent tissue specimens were examined by real-time polymerase chain reaction. miRNA expression levels were also correlated with commonly used clinicopath-ologic features of CRC.  Expression levels of analyzed miRNAs significantly differed among tumors and adjacent non-tumor tissues: miR-21 (p = 0.0001) and miR-31 (p = 0.0006) were upregulated, and miR-143 (p = 0.011) and miR-145 (p = 0.003) were downregulated in tumors. For the first time, a high expression of miR-21 was associated with lymph node positivity (p = 0.025) and the development of distant metastases (p = 0.009) in CRC patients. Thus, expression of miR-21 correlated with CRC clinical stage (p = 0.032). Furthermore, tumors >50 mm in maximal tumor diameter were characterized by lower expression of miR-143 (p = 0.006) and miR-145 (p = 0.003). We found no correlation between analyzed miRNAs and serum levels of carcinoembryonic antigen.  Our results suggest possible roles of miR-21, miR-31, miR-143 and miR-145 in CRC.

title: Role of miR-143 targeting KRAS in colorectal tumorigenesis
abstract: Dysregulated expression of microRNAs (miRNAs) is associated with a variety of diseases, including colorectal cancer. By comparing more than 200 miRNAs in 13 pairs of matched colorectal cancer and normal adjacent tissue samples through qRT-PCR and microarray analysis, we found a widespread disruption of miRNA expression during colorectal tumorigenesis. In particular, among a panel of presumed targets generated by in silico analysis that may interact with these aberrantly expressed miRNAs, KRAS oncogene has been further experimentally validated as the target of miR-143. First, an inverse correlation between KRAS protein and miR-143 in vivo was found. Second, KRAS expression in Lovo cells was significantly abolished by treatment with miR-143 mimic, whereas miR-143 inhibitor increased KRAS protein level. Third, luciferase reporter assay confirmed that miR-143 directly recognize the 3′-untranslated region of KRAS transcripts. Four, Lovo cells treated with miR-143 inhibitor showed a stimulated cell proliferation, whereas miR-143 overexpression had an opposite effect. Finally, inhibition of KRAS expression by miR-143 inhibits constitutive phosphorylation of ERK1/2. Taken together, the present study provides the first evidences that miR-143 is significant in suppressing colorectal cancer cell growth through inhibition of KRAS translation.

title: Clinicopathological Significance of microRNA-31, -143 and -145 Expression in Colorectal Cancer
abstract: We are just beginning to understand how microRNAs (miRNAs) are involved in tumor-related processes in humans. Applying real-time RT-PCR, we investigated the miR-31, miR-143 and miR-145 expression in 98 primary CRC specimens, along with the corresponding normal mucosa specimens, and analyze the relationship of their expression with clinicopathological features. Our results showed the miR-31 expression was up-regulated in CRC compared to normal mucosa (p = 0.001). Furthermore, miR-31 expression was positively related to advanced TNM stage (p = 0.026) and deeper invasion of tumors (p = 0.024). MiR-145 was down-regulated in both colon (p = 0.001) and rectal (p = 0.012) cancer. MiR-143 was only down-regulated in colon cancer (p = 0.023) but not in rectal cancer (p = 0.351). There was no relationship of miR-143 and miR-145 expression with other clinicopathological features (p > 0.05), except that the miR-145 expression was related to cancer site (p = 0.03). In conclusion, the miR-31 overexpression may be involved in the development and progression of CRC. The miR-143 and miR-145 may play a certain role in the development of colon and/or rectal cancers but not in progression of the disease.

title: MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.
abstract: MicroRNAs have potential as diagnostic biomarkers and therapeutic targets in cancer. No study has evaluated the association between microRNA expression patterns and colon cancer prognosis or therapeutic outcome.



To identify microRNA expression patterns associated with colon adenocarcinomas, prognosis, or therapeutic outcome.



MicroRNA microarray expression profiling of tumors and paired nontumorous tissues was performed on a US test cohort of 84 patients with incident colon adenocarcinoma, recruited between 1993 and 2002. We evaluated associations with tumor status, TNM staging, survival prognosis, and response to adjuvant chemotherapy. Associations were validated in a second, independent Chinese cohort of 113 patients recruited between 1991 and 2000, using quantitative reverse transcription polymerase chain reaction assays. The final date of follow-up was December 31, 2005, for the Maryland cohort and August 16, 2004, for the Hong Kong cohort.



MicroRNAs that were differentially expressed in tumors and microRNA expression patterns associated with survival using cancer-specific death as the end point.  Thirty-seven microRNAs were differentially expressed in tumors from the test cohort. Selected for validation were miR-20a, miR-21, miR-106a, miR-181b, and miR-203, and all 5 were enriched in tumors from the validation cohort (P < .001). Higher miR-21 expression was present in adenomas (P = .006) and in tumors with more advanced TNM staging (P < .001). In situ hybridization demonstrated miR-21 to be expressed at high levels in colonic carcinoma cells. The 5-year cancer-specific survival rate was 57.5% for the Maryland cohort and was 49.5% for the Hong Kong cohort. High miR-21 expression was associated with poor survival in both the training (hazard ratio, 2.5; 95% confidence interval, 1.2-5.2) and validation cohorts (hazard ratio, 2.4; 95% confidence interval, 1.4-3.9), independent of clinical covariates, including TNM staging, and was associated with a poor therapeutic outcome.



Expression patterns of microRNAs are systematically altered in colon adenocarcinomas. High miR-21 expression is associated with poor survival and poor therapeutic outcome.

title: Let‐7c functions as a metastasis suppressor by targeting MMP11 and PBX3 in colorectal cancer
abstract: Accumulating evidence shows that microRNAs, functioning as either oncogenes or tumour suppressors by negatively regulating downstream target genes that are actively involved in tumour initiation and progression, may be promising biomarkers and therapy targets. Data mining through a microRNA chip database indicated that let‐7c may be associated with tumour metastasis. Here, we confirmed that down‐regulation of let‐7c in primary cancer tissues was significantly associated with metastases, advanced TNM stages and poor survival of colorectal cancer patients. Moreover, ectopic expression of let‐7c in a highly metastatic Lovo cell line remarkably suppressed cell migration and invasion in vitro by the down‐regulation of K‐RAS, MMP11 and PBX3, as well as tumour growth and metastases in vivo, whereas inhibition of let‐7c in low‐metastatic HT29 cells increased cell motility and invasion by the enhanced gene expression of K‐RAS, MMP11 and PBX3. Interestingly, the luciferase reporters' activities with the 3′‐UTRs of K‐RAS, MMP11 and PBX3 were inhibited significantly by let‐7c. Importantly, rescue experiments involving the over‐expression of these genes without their 3′‐UTRs completely reversed the effects of let‐7c on tumour metastasis, both in vitro and in vivo. Finally, the levels of let‐7c were inversely correlated with those of MMP11 and PBX3, but not with those of K‐RAS. Taken together, these results demonstrate that let‐7c, apart from its tumour growth suppression role, also functions as a tumour metastasis suppressor in colorectal cancer by directly destabilizing the mRNAs of MMP11 and PBX3 at least.

title: NIRF is frequently upregulated in colorectal cancer and its oncogenicity can be suppressed by let-7a microRNA.
abstract: Np95 ICBP90 RING finger (NIRF) is essential for the regulation of cell proliferation and has been implicated in tumorigenesis. However, the role of NIRF in colorectal cancer (CRC) remains unclear. In this study, we demonstrated that NIRF expression was aberrantly increased in CRC tissues and associated with poor overall survival. Bioinformatics analysis indicated that NIRF was a putative target of the microRNA let-7a, which was confirmed by luciferase reporter assay. We then demonstrated in vitro that enforced expression of let-7a, or knockdown of NIRF, led to reduced CRC cell proliferation due to cell cycle arrest at the G0/G1 phase and reduced cell migration. Finally, an in vivo tumorigenicity assay in nude mice showed that synthetic let-7a suppressed NIRF expression and reduced tumor growth. Taken together, our results provide new evidence that NIRF has an oncogenic role in CRC. This opens up the possibility of targeting NIRF and let-7a for CRC therapy.

title: Complementary Strand MicroRNAs Mediate Acquisition of Metastatic Potential in Colonic Adenocarcinoma
abstract: BackgroundAltered expression of specific microRNAs (miRNA) is known to occur during colorectal carcinogenesis. However, little is known about the genome-wide alterations in miRNA expression during the neoplastic progression of primary colorectal cancers.MethodsUsing a miRNA array platform, we evaluated the expression of 668 miRNA in primary colonic adenocarcinomas. Biological functions of selected miRNA were evaluated with in vitro invasion assays.ResultsRNA was extracted for miRNA analysis from 65 primary colon cancers. We identified a seven-miRNA expression signature that differentiated stage I and stage IV primary colon cancers. We then demonstrated this signature was able to discriminate between stage II and III primary colon cancers. Six differentially expressed miRNA were downregulated in association with the development of metastases, and all 7 miRNA were complementary strand miRNA. We transfected HCT-116, a highly invasive colon cancer cell line, with corresponding downregulated miRNA and demonstrated that overexpression of three miRNA (miR200c*, miR143*, and miR424*) significantly abrogated invasive potential.ConclusionWe have identified a seven-miRNA signature that is associated with metastatic potential in the primary tumor. Forced overexpression of three downregulated miRNA resulted in attenuation of in vitro invasion, suggesting direct tumor suppressive function and further supporting the biological importance of complementary strand miRNA.

title: Detection of elevated levels of tumour‐associated microRNAs in serum of patients with diffuse large B‐cell lymphoma
abstract: Circulating nucleic acids have been shown to have potential as non‐invasive diagnostic markers in cancer. We therefore investigated whether microRNAs also have diagnostic utility by comparing levels of tumour‐associated MIRN155 (miR‐155), MIRN210 (miR‐210) and MIRN21 (miR‐21) in serum from diffuse large B‐cell lymphoma (DLBCL) patients (n = 60) with healthy controls (n = 43). Levels were higher in patient than control sera (P = 0·009, 0·02 and 0·04 respectively). Moreover, high MIRN21 expression was associated with relapse‐free survival (P = 0·05). This is the first description of circulating microRNAs and suggests that microRNAs have potential as non‐invasive diagnostic markers for DLBCL and possibly other cancers.

title: Circulating microRNAs as stable blood-based markers for cancer detection
abstract: Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

title: Plasma miR-601 and miR-760 Are Novel Biomarkers for the Early Detection of Colorectal Cancer
abstract: Colorectal cancer (CRC) is a major cause of death worldwide. Sensitive, non-invasive diagnostic screen methods are urgently needed to improve its survival rates. Stable circulating microRNA offers unique opportunities for the early diagnosis of several diseases, including cancers. Our aim has been to find new plasma miRNAs that can be used as biomarkers for the detection of CRC.  According to the results of miRNA profiling performed on pooling plasma samples form 10 CRC patients or 10 healthy controls, a panel of miRNAs (hsa-miR-10a, -19a, -22*, -24, -92a, 125a-5p, -141, -150, -188-3p, -192, -210, -221, -224*, -376a, -425*, -495, -572, -601, -720, -760 and hsa-let-7a, -7e) were deregulated in CRC plasma with fold changes >5. After large scale validation by qRT-PCR performed on another 191 independent individuals (90 CRC, 43 advanced adenoma and 58 healthy participants), we found that the levels of plasma miR-601 and miR-760 were significantly decreased in colorectal neoplasia (carcinomas and advanced adenomas) compared with healthy controls. ROC curve analysis showed that plasma miR-601 and miR-760 were of significant diagnostic value for advanced neoplasia. These two miRNAs together yield an AUC of 0.792 with 83.3% sensitivity and 69.1% specificity for separating CRC from normal controls, and yield an AUC of 0.683 with 72.1% sensitivity and 62.1% specificity in discriminating advanced adenomas from normal controls.

title: Plasma MiR-21: A Potential Diagnostic Marker of Colorectal Cancer
abstract: The main objective of this study was to investigate the potential use of circulating microRNAs (miRNAs) as biomarkers of sporadic colorectal cancer (CRC). CRC, a leading cause of death, is curable if detected early. There is an unmet need for an accurate, noninvasive biomarker of CRC. MiRNAs are non–protein-coding RNAs regulating gene expression that play a role in CRC development. Levels of 380 miRNAs were determined using microfluidic array technology (Applied Biosystems) in a “training” set of 30 CRC patients from whom cancer and adjacent normal tissue were collected. The 4 most dysregulated miRNAs (P < 0.05, false discovery rate (FDR): 10%) were then validated in a second blinded “test” set of 16 CRC patients from whom cancer and normal adjacent tissue had been collected. Validated tissue miRNAs were then evaluated in a plasma “test” set consisting of 30 CRC patients and 30 individuals without CRC. The most dysregulated tissue miRNAs were then validated in an independent new plasma test set consisting of 20 CRC patients with 20 age-, -, and race-matched subjects without CRC. Nineteen of 380 miRNAs were dysregulated in CRC tissue in the tissue “training” set (P < 0.05, FDR: 10%). The 2 most upregulated (miR-31; miR-135b) and most downregulated (miR-1; miR-133a) miRNAs identified CRC in our “test” set with 100% sensitivity and 80% specificity. MiR-31 was more upregulated in stages III and IV compared with stages I and II (P < 0.05). In the “plasma” group, miR-21 differentiated CRC patients from controls with 90% specificity and sensitivity. Plasma miRNAs provide reliable and noninvasive markers for CRC. Plasma miR-21 warrants study in larger cohorts. It seems uniquely promising as a plasma biomarker for CRC.

title: MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.
abstract: Most ovarian cancer patients are diagnosed at an advanced stage (67%) and prospects for significant improvement in survival reside in early diagnosis. While expression patterns of a recently identified biomarker family, microRNA, appear to be characteristic of tumor type and developmental origin, microRNA profiling has been limited to tissue specimens. Tumors actively release exosomes into the peripheral circulation and we now demonstrate the association of microRNAs with circulating tumor-derived exosomes.



Circulating tumor exosomes were isolated using a modified MACS procedure with anti-EpCAM. Initially, microRNA profiles of ovarian tumors were compared to those of tumor exosomes isolated from the same patients. Levels of 8 microRNAs (miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205 and miR-214) previously demonstrated as diagnostic, were compared in exosomes isolated from sera specimens of women with benign disease and various stages of ovarian cancer.



MicroRNA from ovarian tumor cells and exosomes from the same patients were positive for 218 of 467 mature microRNAs analyzed. The levels of the 8 specific microRNAs were similar between cellular and exosomal microRNAs (exhibiting correlations from 0.71 to 0.90). While EpCAM-positive exosomes were detectable in both patients with benign ovarian disease and ovarian cancer, exosomal microRNA from ovarian cancer patients exhibited similar profiles, which were significantly distinct from profiles observed in benign disease. Exosomal microRNA could not be detected in normal controls.



These results suggest that microRNA profiling of circulating tumor exosomes could potentially be used as surrogate diagnostic markers for biopsy profiling, extending its utility to screening asymptomatic populations.

title: MicroRNA Expression Profiling of Exfoliated Colonocytes Isolated from Feces for Colorectal Cancer Screening
abstract: To reduce the colorectal cancer (CRC) mortality rate, we have reported several CRC screening methods using colonocytes isolated from feces. Expression analysis of oncogenic microRNA (miRNA) in peripheral blood was recently reported for CRC detection. In the present study, we conducted miRNA expression analysis of exfoliated colonocytes isolated from feces for CRC screening. Two hundred six CRC patients and 134 healthy volunteers were enrolled in the study. miRNA expression of the miR-17-92 cluster, miR-21, and miR-135 in colonocytes isolated from feces as well as frozen tissues was analyzed by quantitative real-time PCR. The expression of the miR-17-92 cluster, miR-21, and miR-135 was significantly higher in CRC tissues compared with normal tissues. The exfoliated colonocytes of 197 CRC patients and 119 healthy volunteers were analyzed because of the presence of sufficient miRNA concentration. miR-21 expression did not differ significantly between CRC patients and healthy volunteers (P = 0.6). The expression of miR-17-92 cluster and miR-135 was significantly higher in CRC patients than in healthy volunteers (P < 0.0001). The overall sensitivity and specificity by using miRNA expression was 74.1% (146/197; 95% confidence interval, 67.4-80.1) and 79.0% (94/119; 95% confidence interval, 70.6-85.9), respectively. Sensitivity was dependent only on tumor location (P = 0.0001). miRNA was relatively well conserved in exfoliated colonocytes from feces both of CRC patients and healthy volunteers. miRNA expression analysis of the isolated colonocytes may be a useful method for CRC screening. Furthermore, oncogenic miRNA highly expressed in CRC should be investigated for CRC screening tests in the future.

title: Differential expression of miR-144* as a novel fecal-based diagnostic marker for colorectal cancer
abstract: BackgroundMicroRNAs (miRNA) are tiny, noncoding, small, endogenous RNAs that play major roles in neoplastic transformation and could therefore offer a better quantitative and noninvasive method for the diagnosis and prognosis of colorectal cancer (CRC) using feces. In the present study, we screened feces for 648 miRNAs and analyzed the role of miR-144* as a potential CRC diagnostic marker.MethodsFecal miRNA expression was profiled with RT-pre-amplification-qPCR, and the stability was determined using both endogenous and exogenous miRNA by RT-qPCR. ROC analysis was performed to enhance the diagnosing power of the CRC patients’ fecal specimens.ResultsWe detected 39% of all the miRNAs screened in feces. Endogenous miRNAs are more stable over time and temperature, while exogenous miRNAs degraded rapidly. miR-144* was overexpressed in feces, suggesting that it could be a potent candidate diagnostic marker for CRC detection, with a sensitivity of 74% and a specificity of 87% (n = 75, p < 0.0001). Moreover, RT-qPCR analysis showed that miR-144* was also overexpressed in paired CRC tissues, thus suggesting its possible utilization as a diagnostic marker.ConclusionsWe demonstrated that miRNAs are stable in the fecal microenvironment, and that, among them, miR-144* represents a novel fecal-based diagnostic marker for CRC screening. Nevertheless, our data need to be validated in a large cohort of subjects.

title: Down-regulation of fecal miR-143 and miR-145 as potential markers for colorectal cancer.
abstract: To detect 4 MicroRNA (miRNA) in the stool samples of colorectal cancer (CRC) patients to determine whether these miRNAs could be biomarkers in CRC screening or treatment.



A retrospective comparison study was carried out in the Department of Colorectal Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China from September 2009 to March 2011. We detected 4 miRNAs (miR-143, miR-145, miR-21, and miR-106a) in the stool samples of 38 CRC patients and 13 healthy individuals. Total RNA from the stool samples was extracted using the EZNA TM stool RNA kit R6828-01. The miRNA quantification was carried out using TaqMan miRNA assays and the TaqMan Gene Expression Master Mix.



The expression levels of miR-143 and miR-145 in the stool of the CRC patients were lower than in those of the healthy persons (p<0.005, median of 2-∆∆ct). No statistically significant difference was found in the expression levels of both miR-21 and miR-106a between the stool of CRC patients and those of the healthy persons (p>0.05).



The detection of fecal miRNAs is a potential method for CRC diagnosis or screening. Particularly, the down-regulation of fecal miR-143 and miR-145 could be a potential marker for CRC.

title: Detection of miR-92a and miR-21 in stool samples as potential screening biomarkers for colorectal cancer and polyps
abstract: The detection of molecular markers in stool samples is a potential strategy for colorectal cancer (CRC) screening. This study evaluated the feasibility of detecting miR-21 and miR-92a in stool samples of patients with CRC or polyps.  The reproducibility of detection and stability of stool-based microRNA were evaluated. Stool samples were collected from 88 patients with CRC, 57 patients with colorectal polyps and 101 healthy controls. MiRNA levels in CRC tissues and stool samples were detected by real-time quantitative reverse transcription PCR. Stool miR-21 and miR-92a levels were compared before and after the removal of tumour or advanced adenoma.  The study demonstrated that stool-based miRNA were stable with highly reproducible detection. The expression of miR-21 and miR-92a was significantly higher in CRC tissues compared with their adjacent normal tissues (p<0.0001). Patients with CRC had a significantly higher stool miR-21 level (p<0.01) and miR-92a level (p<0.0001) compared with normal controls. Stool miR-92a, but not miR-21, was significantly higher in patients with polyps than in controls (p<0.0001). At a cut-off value of 435 copies/ng of stool RNA, miR-92a had a sensitivity of 71.6% and 56.1% for CRC and polyp, respectively, and a specificity of 73.3%. In addition, the stool miR-92a level demonstrated a higher sensitivity for distal CRC than proximal CRC (p<0.05), and a higher sensitivity for advanced adenoma than minor polyps (p<0.05). Removal of tumour resulted in reduced stool miR-21 and miR-92a levels (p<0.01), and the removal of advanced adenoma resulted in a reduction of the stool miR-92a level (p<0.05).  Stool miRNA are useful for screening CRC and polyps.

title: MicroRNAs: predictors and modifiers of chemo- and radiotherapy in different tumour types.
abstract: MicroRNAs (miRNAs) represent a class of naturally occurring small non-coding RNA molecules. They regulate gene expression at the post-transcriptional level and control thereby cellular mechanisms including developmental transitions, organ morphology, apoptosis and cell proliferation. As might be expected from molecules with these roles, miRNAs are involved in cancer development, and deregulation of several miRNAs has been found in various cancer types. Some miRNAs modulate expression of known oncogenes or tumour suppressor genes whereas others function as so called onco-miRs or tumour-suppressor-miRs. Recently, miRNAs have been studied as potential diagnostic or therapeutic targets in cancer treatment. There is increasing interest in an association between miRNA expression in tumours and chemo- and radiosensitivity, both with regards to predicting or modulating sensitivity. And indeed, different miRNAs have been found to predict sensitivity to anticancer treatment: miR-30c, miR-130a and miR-335 are downregulated in various chemoresistant cell lines, hsa-Let-7g and hsa-miR-181b are strongly associated with response to 5-fluorouracil-based antimetabolite S-1. In addition, several miRNAs were shown to influence sensitivity to chemo- or radiotherapy: miRNAs of the Let-7 family induced radiosensitivity in vitro/in vivo, inhibition of miR-21 and miR-200b increased sensitivity to gemcitabine in cholangiocarcinoma cell lines, and restoration of miR-34 in p53-deficient human gastric cancer cells induced chemosensitisation. This article summarises the current literature describing the impact of miRNAs on prediction and modification of anticancer treatment including the possible intracellular pathways involved in these processes.

title: miR-192/miR-215 Influence 5-Fluorouracil Resistance through Cell Cycle-Mediated Mechanisms Complementary to Its Post-transcriptional Thymidilate Synthase Regulation
abstract: Thymidylate synthase (TYMS) is a target of the most widely used chemotherapeutic agents against gastrointestinal malignancies, the fluoropyrimidine-based therapy. TYMS expression levels have been identified as predictive biomarkers for 5-fluoruracil (FU) response in colorectal cancer, but their clinical utility remains controversial. The complexity of fluoropyrimidine response must require more mechanisms that currently have not been completely elucidated. In this context, microRNAs (miRNA) may play a role in modulating chemosensitivity. By carrying out an in silico analysis coupled to experimental validation, we detected that miR-192 and miR-215 target TYMS expression in colorectal cancer cell lines. However, downregulation of TYMS by these miRNAs does not sensitize colorectal cancer cell lines to FU treatment. The overexpression of miR-192/215 significantly reduces cell proliferation by targeting cell cycle progression. This effect was partially associated with p53 status, because reduction of cell proliferation and cell cycle arrest was associated with p21 and p27 induction. The decrease of S-phase cells by these miRNAs mitigates the effects of S phase–specific drugs and suggests that other mechanisms different from TYMS overexpression are essential to direct FU resistance. Finally, ectopic expression of miR-192/215 might have stronger impact to predict FU response than TYMS inhibition. Prospective studies to elucidate the role of these miRNAs as predictive biomarkers to FU are necessary.

title: miR-181a is associated with poor clinical outcome in patients with colorectal cancer treated with EGFR inhibitor
abstract: miR-181a expression is frequently altered in different types of cancer. Members of the Wnt/β-catenin signalling pathway, which is commonly altered in colorectal cancer (CRC), have been reported as molecular interaction partners of miR-181. However, the role of miR-181a expression in CRC and its ability to predict survival and response to agents targeting the epidermal growth factor receptor (EGFR) have not been explored yet.  In this study, we analysed 80 patients with wild type KRAS CRC undergoing treatment with the EGFR-targeting monoclonal antibodies cetuximab and panitumumab for metastatic CRC. The KRAS mutational status was determined by pyrosequencing and miR-181a expression was measured by quantitative RT-PCR in CRC tumour tissue and corresponding non-neoplastic colon tissue. The microRNA expression levels were correlated with clinicopathological characteristics. Cancer-specific survival was calculated by univariate and multivariate analyses, and progression-free survival (PFS) during treatment with EGFR-targeting agents was also evaluated.  A low miR-181a expression level was associated with poor differentiation of CRC (p=0.04). A Kaplan-Meier curve showed a decreased survival time for patients with low miR-181a expression (p=0.019). Low miR-181a expression was furthermore associated with poor PFS (p=0.015).  In conclusion, our data suggest that the miR-181a expression level is associated with poor survival in patients with CRC. Furthermore, miR-181a expression might predict PFS in EGFR-targeted therapy.

title: Role of miR‐200 family members in survival of colorectal cancer patients treated with fluoropyrimidines
abstract: Surgery is the standard treatment for colorectal cancer (CRC), and adjuvant chemotherapy has been shown to be effective in stage III but less so in stage II. We have analyzed the expression of the miR‐200 family in tissue samples from resected CRC patients and correlated our findings with survival to adjuvant treatment with fluoropyrimidines.

title: Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy?
abstract: MicroRNAs (miRNAs) were discovered more than a decade ago as noncoding, single-stranded small RNAs (∼22 nucleotides) that control the timed gene expression pattern in Caenorhabditis elegans life cycle. A number of these evolutionarily conserved, endogenous miRNAs have been shown to regulate mammalian cell growth, differentiation and apoptosis. miRNAs are multispecific by nature. The individual miRNA is capable of modulating the expression of a network of mRNAs that it binds by imperfect sequence complementarity. Human cancers commonly exhibit an altered expression profile of miRNAs with oncogenic (miR-21, miR-106a and miR-155) or tumor-suppressive (let-7, miR-15a/16, miR-34a and miR-143/145) activity. As consistent with the natural function of miRNAs in specifying cellular phenotype, miRNA-based cancer gene therapy offers the theoretical appeal of targeting multiple gene networks that are controlled by a single, aberrantly expressed miRNA. Reconstitution of tumor-suppressive miRNA, or sequence-specific knockdown of oncogenic miRNAs by ‘antagomirs,’ has produced favorable antitumor outcomes in experimental models. We discuss pending issues that need to be resolved prior to the consideration of miRNA-based experimental cancer gene therapy. These include the need for definitive mRNA target validation, our incomplete understanding of rate-limiting cellular components that impact the efficiency of this posttranscriptional gene-silencing phenomenon, the possibility for nonspecific immune activation and the lack of a defined, optimal mode of delivery.

title: Insights into the diverse roles of miR-205 in human cancers.
abstract: The recent discovery of tiny microRNAs (miRNAs) has brought about awareness of a new class of regulators of diverse pathways in many physiological and pathological processes, such as tumorigenesis. They modulate gene expression by targeting plethora of mRNAs, mostly reducing the protein yield of a targeted mRNA. With accumulation of information on characteristics of miR-205, complex and in some cases converse roles of miR-205 in tumor initiation, progression and metastasis are emerging. miR-205 acts either as an oncogene via facilitating tumor initiation and proliferation, or in some cases as a tumor suppressor through inhibiting proliferation and invasion. The aim of this review is to discuss miR-205 roles in different types of cancers. Given the critical effects of deregulated miR-205 on processes involved in tumorigenesis, they hold potential as novel therapeutic targets and biomarkers.

title: miR-133b regulates the MET proto-oncogene and inhibits the growth of colorectal cancer cells in vitro and in vivo
abstract: Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRs) are single-stranded, noncoding RNAs that are important in many biological processes. Although the oncogenic and tumor-suppressive functions of several miRs have been characterized, their precise biological roles remain largely unexplored. In the present study, the role of miR-133b was identified in the regulation of CRC cell proliferation and apoptosis. miR-133b expression was shown to be greatly downregulated in human CRC cells compared to normal colon cells. Downregulation of miR-133b expression was also significant in six of eight human CRC tissues compared with adjacent normal tissues. In the CRC cell lines SW-620 and HT-29, ectopic expression of miR-133b potently affected tumor cell proliferation and apoptosis in vitro and in vivo by direct targeting of the receptor tyrosine kinase MET. Transfection of SW-620 and HT-29 cells with miR-133b significantly suppressed a luciferase-reporter containing the MET-3′-untranslated region. Taken together, these results provide evidence that miR-133b regulated tumor cell proliferation and apoptosis through modulation of the MET signaling pathway.

title: miR‐137 targets Cdc42 expression, induces cell cycle G1 arrest and inhibits invasion in colorectal cancer cells
abstract: miRNAs have emerged as post‐transcriptional regulators that are critically involved in the pathogenesis of a number of human cancers. Cdc42, one of the best characterized members of the Rho GTPase family, is found to be up‐regulated in several types of human tumors and has been implicated in cancer initiation and progression. In the present study, we have identified miR‐137 as a potential regulator of Cdc42 expression. A bioinformatics search revealed a putative target‐site for miR‐137 within the Cdc42 3′ UTR at nt 792–798, which is highly conserved across different species. Expression of miR‐137 in colorectal cancer cell lines was found inversely correlated with Cdc42 expression. miR‐137 could significantly suppress Cdc42 3′ UTR luciferase‐reporter activity, and this effect was not detectable when the putative 3′ UTR target‐site was mutated. Consistent with the results of the reporter assay, ectopic expression of miR‐137 reduced both mRNA and protein expression levels of Cdc42 and mimicked the effect of Cdc42 knockdown in inhibiting proliferation, inducing G1 cell cycle arrest, and blocking invasion of the colorectal cancer cells, whereas anti‐miR‐137 expression led to the opposite effect. Furthermore, expression of miR‐137 suppressed the immediate downstream effector of Cdc42, PAK signaling. Our results suggest that miR‐137 may have a tumor suppressor function by directly targeting Cdc42 to inhibit the proliferation and invasion activities of colorectal cancer cells. They raise an interesting possibility that Cdc42 activity and function can be controlled by miRNAs in addition to the classic regulators such as guanine nucleotide exchange factors and GTPase‐activating proteins.

title: Putative tumor suppressor miR‐145 inhibits colon cancer cell growth by targeting oncogene friend leukemia virus integration 1 gene
abstract: Tumor suppressor microRNA miR‐145 is commonly down‐regulated in colon carcinoma tissues, but its specific role in tumors remains unknown.

title: Micro RNA 145 Targets the Insulin Receptor Substrate-1 and Inhibits the Growth of Colon Cancer Cells*
abstract: The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3′-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3′-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.

title: MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer
abstract: MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes, including some involved in cancer development. In this study, we investigated the possible role of miR-143 in colorectal cancer (Expression levels of human mature miRNAs were examined using real-time PCR-based expression arrays on paired colorectal carcinomas and adjacent non-cancerous colonic tissues. The downregulation of miR-143 was further evaluated in colon cancer cell lines and in paired CRC and adjacent non-cancerous colonic tissues by qRT–PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA–target association.Both real-time PCR-based expression arrays and qRT–PCR showed that miR-143 was frequently downregulated in 87.5% (35 of 40) of colorectal carcinoma tissues compared with their adjacent non-cancerous colonic tissues. Using in silico predictions, DNA methyltranferase 3A (DNMT3A) was defined as a potential target of miR-143. Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels. DNMT3A was shown to be a direct target of miR-143 by luciferase reporter assay. Furthermore, the miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in CRC tissues.Our findings suggest that miR-143 regulates DNMT3A in CRC. These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

title: Functional screening identifies a microRNA, miR‐491 that induces apoptosis by targeting Bcl‐XL in colorectal cancer cells
abstract: MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain‐of‐function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD‐1). We discovered a number of miRNAs that increased or decreased cell viability in DLD‐1. They included known oncogenic miRNAs such as miR‐372 and miR‐373, and tumor suppressive miRNAs such as miR‐124a, but also some for which this information was novel. Among them, miR‐491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl‐XL was a direct target of miR‐491, and its silencing contributed to miR‐491‐induced apoptosis. Moreover, treatment of miR‐491 suppressed in vivo tumor growth of DLD‐1 in nude mice. Our study provides a new regulation of Bcl‐XL by miR‐491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl‐XL.

title: Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening
abstract: MicroRNAs (miRNAs) have been shown to offer great potential in the diagnosis of cancer. We investigated whether plasma miRNAs could discriminate between patients with and without colorectal cancer (CRC).  This study was divided into three phases: (1) marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of five patients with CRC, along with plasma from five healthy individuals as controls; (2) marker selection and validation by real-time quantitative RT-PCR on a small set of plasma; and (3) independent validation on a large set of plasma from 90 patients with CRC, 20 patients with gastric cancer, 20 patients with inflammatory bowel disease (IBD) and 50 healthy controls.  Of the panel of 95 miRNAs analysed, five were upregulated both in plasma and tissue samples. All the five miRNAs were validated on the plasma of 25 patients with CRC and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in the patients with CRC (p<0.0005). The plasma levels of these markers were significantly reduced after surgery in 10 patients with CRC (p<0.05). Further validation with an independent set of plasma samples (n = 180) indicated that miR-92 differentiates CRC from gastric cancer, IBD and normal subjects. This marker yielded a receiver operating characteristic curve area of 88.5%. At a cut-off of 240 (relative expression in comparison to RNU6B snRNA), the sensitivity was 89% and the specificity was 70% in discriminating CRC from control subjects.  MiR-92 is significantly elevated in plasma of patients with CRC and can be a potential non-invasive molecular marker for CRC screening.

title: Identification of serum microRNA profiles in colon cancer
abstract: microRNAs (miRNAs) exist in blood in an apparently stable form. We have explored whether serum miRNAs can be used as non-invasive early biomarkers of colon cancer.Serum samples from 30 patients with colon cancer stage IV and 10 healthy controls were examined for the expression of 375 cancer-relevant miRNAs. Based on the miRNA profile in this study, 34 selected miRNAs were measured in serum from 40 patients with stage I–II colon cancer and from 10 additional controls.Twenty miRNAs were differentially expressed in serum from stage IV patients compared with controls (P<0.01). Unsupervised clustering revealed four subgroups; one corresponding mostly to the control group and the three others to the patient groups. Of the 34 miRNAs measured in the follow-up study of stage I–II patients, 21 showed concordant expression between stage IV and stage I–II patient. Based on the profiles of these 21 miRNAs, a supervised linear regression analysis (Partial Least Squares Regression) was performed. Using this model we correctly assigned stage I–II colon cancer patients based on miRNA profiles of stage IV patients.Serum miRNA expression profiling may be utilised in early detection of colon cancer.

title: Circulating miR‐221 directly amplified from plasma is a potential diagnostic and prognostic marker of colorectal cancer and is correlated with p53 expression
abstract: Circulating miRNAs exist in serum and plasma and they can be used as a potential noninvasive molecular marker for colorectal cancer (CRC) diagnosis. The present study was to test the availability of direct amplification of miRNAs from plasma without RNA extraction, and to evaluate its clinical application value in CRC.

title: Identification and Validation of Reference Genes for qPCR Detection of Serum microRNAs in Colorectal Adenocarcinoma Patients
abstract: Serum microRNAs (miRNAs) have become a highlighted research hotspot, especially for their great potential as a novel promising non-invasive biomarker in cancer diagnosis. The most frequently used approach for serum miRNAs detection is quantitative real time polymerase chain reaction (qPCR). In order to obtain reliable qPCR data of miRNAs expression, the use of reference genes as endogenous control is undoubtly necessary. However, no systematic evaluation and validation of reference genes for normalizing qPCR analysis of serum miRNAs has been reported in colorectal adenocarcinoma. We firstly profiled pooled serum of colorectal adenocarcinoma, colorectal adenoma and healthy controls and selected a list of 13 miRNAs as candidate reference genes. U6 snRNA (U6) and above-mentioned 13 miRNAs were included in further confirmation by qPCR. As a result, 5 miRNAs (miR-151a-3p, miR-4446-3p, miR-221-3p, miR-93-5p and miR-3184-3p) were not detected in all samples and 2 miRNAs (miR-197-3p and miR-26a-5p) were relatively low with median Cq more than 35, and were excluded from further stability analysis. Then variable stability of other 6 miRNAs (miR-103b, miR-484, miR-16-5p, miR-3615, miR-18a-3p and miR-191-5p) and U6 were evaluated using two algorithms: geNorm and NormFinder which both identified miR-191-5p as the most stably expressed reference gene and selected miR-191-5p and U6 as the most stable pair of reference genes. After validating in an independent large cohorts and selecting miR-92a-3p as target miRNA to evaluate the effect of reference gene, we propose that combination of miR-191-5p and U6 could be used as reference genes for serum microRNAs qPCR data in colorectal adenocarcinoma, colorectal adenoma and healthy controls.