Source: http://www.patentsencyclopedia.com/app/20120094374
Timestamp: 2017-10-20 03:57:31
Document Index: 640629924

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'application No. 60']

Patent application number: 20120094374
18. An isolated nucleic acid of 18-200 nucleotides in length comprising a sequence selected from the group consisting of: (a) SEQ ID NO: 908; (b) a DNA encoding the nucleic acid of (a), wherein the DNA is identical in length to (a); (c) a sequence at least 80% identical to (a) or (b); and (d) the complement of (a), (b) or (c), wherein the complement is identical in length to the nucleic acid of (a), (b), or (c).
19. An isolated nucleic acid of 18-100 nucleotides in length comprising a sequence selected from the group consisting of: (a) SEQ ID NO: 8807 or a fragment thereof wherein the fragment is 18-22 nucleotides in length; (b) a DNA encoding the nucleic acid of (a), wherein the DNA is identical in length to (a); (c) a sequence at least 80% identical to (a) or (b); and (d) the complement of (a),(b) or (c), wherein the complement is identical in length to the nucleic acid of (a), (b), or (c).
[0001] This is a divisional of U.S. patent application Ser. No. 11/130,645, filed May 16, 2005, which is a continuation of International Application No. PCT/U.S. 2005/16986, filed May 14, 2005, which is a continuation-in-part of U.S. application Ser. No. 10/709,572 filed May 14, 2004, and U.S. application Ser. No. 10/709,577, filed May 14, 2004, and which claims the benefit of U.S. Provisional Application No. 60/666,340, filed Mar. 30, 2005, U.S. Provisional Application No. 60/665,094, filed Mar. 25, 2005, U.S. Provisional Application No. 60/662,742, filed March 17, 2005, U.S. Provisional Application No. 60/593,329, filed Jan. 6, 2005, U.S. Provisional Application No. 60/593,081, filed Dec. 8, 2004, U.S. Provisional Application No. 60/522,860, filed Nov. 15, 2004, U.S. Provisional Application No. 60/522,457, filed Oct. 4, 2004, U.S. Provisional Application No. 60/522,452, filed Oct. 3, 2004, and U.S. Provisional Application No. 60/522,449, filed Oct. 3, 2004, the contents of which are incorporated herein by reference.
REFERENCES TO THE SEQUENCE LISTING AND COMPUTER PORGRAM LISTING APPENDICES
[0003] Applicant hereby makes reference to the sequence listing and computer program listing appendices that were submitted on compact disc. The sequence listing consists of a filed named "SeqList.txt," (168,151 bytes), created on Mar. 5, 2010. The computer program listing appendix consists of the following files: "Table 1.bat" (968 KB), "Table 2.bat" (1327 KB), "Table 3.bat" (9 KB), "Table 4.bat" (10,857 KB), "Table 5.bat" (986 KB), "Table 6.bat" (38 KB), "Table 7.bat" (2 KB), "Table 8.bat" (4 KB), "Table 9.bat" (190 KB), "Table 10--001.bat" (716,801 KB), "Table 10--002.bat" (716,801 KB), "Table 10--003.bat" (105,669 KB), "Table 11A.bat" (634,769 KB), "Table 11B.bat" (634,770 KB), "Table 11C.bat " (113,878 KB), "Table 12.bat" (585,921 KB), "Table 13A.bat" (634,767 KB), "Table 13B.bat" (51,584 KB), "Table 14.bat" (75 KB), "Table 15.bat" (68 KB), "Table 16.bat" (774 KB), and "Table 17.bat" (3183 KB), all of which were created on Feb. 17, 2010. The sequence listing and computer program listing appendices are all incorporated herein by reference.
[0018] FIG. 2 shows a schematic illustration of the MC19cluster on 19q13.42. Panel A shows the ˜500,000bp region of chromosome 19, from 58,580,001 to 59,080,000 (according to the May 2004 USCS assembly), in which the cluster is located including the neighboring protein-coding genes. The MC19-1 cluster is indicated by a rectangle. Mir-371, mir-372, and mir-373 are indicted by lines. Protein coding genes flanking the cluster are represented by large arrow-heads. Panel B shows a detailed structure of the MC19-1 miRNA cluster. A region of ˜102,000bp, from 58,860,001 to 58,962,000 (according to the May 2004 USCS assembly), is presented. MiRNA precursors are represented by a black bars. It should be noted that all miRNAs are at the same orientation from left to right. Shaded areas around miRNA precursors represent repeating units in which the precursor is embedded. The location of mir-371, mir-372, and mir-373, is also presented.
[0019] FIG. 3 is a graphical representation of multiple sequence alignment of 35 human repeat units at distinct size of ˜690 nt (A) and 26 chimpanzees repeat units (B). The graph was generated by calculating a similarity score for each position in the alignment with an averaging sliding window of 10 nt (Maximum score -1, minimum score -0). The repeat unit sequences were aligned by ClustalW program. Each position of the resulting alignment was assigned a score which represented the degree of similarity at this position. The region containing the miRNA precursors is bordered by vertical lines. The exact location of the mature miRNAs derived from the 5' stems (5 p) and 3' stems (3 p) of the precursors is indicted by vertical lines.
[0020] FIG. 4 shows sequence alignments of the 43 A-type pre-miRNAs of the MC19-1 cluster. Panel A shows the multiple sequence alignment with the Position of the mature miRNAs marked by a frame. The consensus sequence is shown at the bottom. Conserved nucleotides are colored as follows: black-100%, dark grey-80% to 99%, and clear grey-60% to 79%. Panel B shows alignments of consensus mature A-type miRNAs with the upstream human cluster of mir-371, mir-372, miR-373. Panel C shows alignments of consensus mature A-type miRNAs with the hsa-mir-371-373 mouse orthologous cluster. The miRNAs hsa-miR-A1 through hsa-miR-A43 of panel A have SEQ ID NOs: 760625 through 760667, respectively. In panel B, the following miRNAs have the following sequences: hsa-miR-371, 3p (SEQ ID NO: 760668), hsa-miR-372, 3p (SEQ ID NO: 760669), hsa-miR-373, 3p (SEQ ID NO: 760670), hsa-miR-A-3p (consensus)(SEQ ID NO: 760671), hsa-miR-373-5-p (SEQ ID NO: 760672), hsa-miR-A-5p (consensus)(SEQ ID NO: 760678). In panel C, the following miRNAs have the following sequences: hsa-miR-302a (SEQ ID NO: 760673), hsa-miR-302b (SEQ ID NO: 760674), hsa-miR-302c (SEQ ID NO: 760675), hsa-miR-302d (SEQ ID NO: 760676), and hsa-miR-A-3p (consensus)(SEQ ID NO: 760677).
[0023] Before the present compounds, products and compositions and methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. [0024] a. animal
[0025] "Animal" as used herein may mean fish, amphibians, reptiles, birds, and mammals, such as mice, rats, rabbits, goats, cats, dogs, cows, apes and humans. [0026] b. attached
[0027] "Attached" or "immobilized" as used herein to refer to a probe and a solid support may mean that the binding between the probe and the solid support is sufficient to be stable under conditions of binding, washing, analysis, and removal. The binding may be covalent or non-covalent. Covalent bonds may be formed directly between the probe and the solid support or may be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Non-covalent binding may be one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non-covalent binding of a biotinylated probe to the streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions. [0028] c. biological sample
[0029] "Biological sample" as used herein may mean a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue isolated from animals. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A biological sample may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, such as those having treatment or outcome history, may also be used. [0030] d. complement
[0031] "Complement" or "complementary" as used herein may mean Watson-Crick or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. [0032] e. differential expression
[0033] "Differential expression" may mean qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue. Thus, a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus disease tissue. Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states. A qualitatively regulated gene will exhibit an expression pattern within a state or cell type which may be detectable by standard techniques. Some genes will be expressed in one state or cell type, but not in both. Alternatively, the difference in expression may be quantitative, e.g., in that expression is modulated, either up-regulated, resulting in an increased amount of transcript, or down-regulated, resulting in a decreased amount of transcript. The degree to which expression differs need only be large enough to quantify via standard characterization techniques such as expression arrays, quantitative reverse transcriptase PCR, northern analysis, and RNase protection. [0034] f. gene
[0035] "Gene" used herein may be a genomic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5'- and 3'-untranslated sequences). The coding region of a gene may be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene may also be an mRNA or cDNA corresponding to the coding regions (e.g., exons and miRNA) optionally comprising 5'- or 3'-untranslated sequences linked thereto. A gene may also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3'-untranslated sequences linked thereto. [0036] g. host cell
[0037] "Host cell" used herein may be a naturally occurring cell or a transformed cell that contains a vector and supports the replication of the vector. Host cells may be cultured cells, explants, cells in vivo, and the like. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, such as CHO, HeLa. [0038] h. identity
[0039] "Identical" or "identity" as used herein in the context of two or more nucleic acids or polypeptide sequences, may mean that the sequences have a specified percentage of nucleotides or amino acids that are the same over a specified region. The percentage may be calculated by comparing optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces staggered end and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) are considered equivalent. Identity may be performed manually or by using computer sequence algorithm such as BLAST or BLAST 2.0. [0040] i. label
[0041] "Label" as used herein may mean a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable. A label may be incorporated into nucleic acids and proteins at any position. [0042] j. nucleic acid
[0045] A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated by reference. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids. The modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2'-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. [0046] k. operably linked
[0047] "Operably linked" used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function. [0048] 1. probe
[0049] "Probe" as used herein may mean an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. A probe may be single stranded or partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. Probes may be directly labeled or indirectly labeled such as with biotin to which a streptavidin complex may later bind. [0050] m. promoter
[0051] "Promoter" as used herein may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter. [0052] n. selectable marker
[0053] "Selectable marker" used herein may mean any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct. Representative examples of selectable markers include the ampicillin-resistance gene (Ampr), tetracycline-resistance gene (TO, bacterial kanamycin-resistance gene (Kanr), zeocin resistance gene, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gene (nptII), hygromycin-resistance gene, beta-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein-encoding gene and luciferase gene. [0054] o. stringent hybridization conditions
[0055] "Stringent hybridization conditions" used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2x SSC, and 0.1% SDS at 65° C. [0056] p. substantially complementary
[0057] "Substantially complementary" used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions. [0058] q. substantially identical
[0059] "Substantially identical" used herein may mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence. [0060] r. target
[0061] "Target" as used herein may mean a polynucleotide that may be bound by one or more probes under stringent hybridization conditions. [0062] s. terminator
[0063] "Terminator" used herein may mean a sequence at the end of a transcriptional unit which signals termination of transcription. A terminator may be a 3'-non-translated DNA sequence containing a polyadenylation signal, which may facilitate the addition of polyadenylate sequences to the 3'-end of a primary transcript. A terminator may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. Representative examples of terminators include the SV40 polyadenylation signal, HSV TK polyadenylation signal, CYC1 terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, rho-independent E. coli terminators, and the lacZ alpha terminator. [0064] t. Vector
[0069] Although initially present as a double-stranded species with miRNA*, the miRNA may eventually become incorporated as single-stranded RNAs into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Various proteins can form the RISC, which can lead to variability in specifity for miRNA/miRNA* duplexes, binding site of the target gene, activity of miRNA (repress or activate), which strand of the miRNA/miRNA* duplex is loaded in to the RISC.
[0072] A number of studies have looked at the base-pairing requirement between miRNA and its mRNA target for achieving efficient inhibition of translation (reviewed by Bartel 2004, Cell 116-281). In mammalian cells, the first 8 nucleotides of the miRNA may be important (Doench & Sharp 2004 GenesDev 2004-504). However, other parts of the microRNA may also participate in mRNA binding. Moreover, sufficient base pairing at the 3' can compensate for insufficient pairing at the 5' (Brennecke at al, 2005 PLoS 3-e85). Computation studies, analyzing miRNA binding on whole genomes have suggested a specific role for bases 2-7 at the 5' of the miRNA in target binding but the role of the first nucleotide, found usually to be "A" was also recognized (Lewis et at 2005 Cell 120-15). Similarly, nucleotides 1-7 or 2-8 were used to identify and validate targets by Krek et al (2005, Nat Genet 37-495).
[0077] The nucleic acid may have a length of from 10 to 100 nucleotides. The nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80 or 90 nucleotides. The nucleic acid may be synthesized or expressed in a cell (in vitro or in vivo) using a synthetic gene described below. The nucleic acid may be synthesized as a single strand molecule and hybridized to a substantially complementary nucleic acid to form a duplex, which is considered a nucleic acid of the invention. The nucleic acid may be introduced to a cell, tissue or organ in a single- or double-stranded form or capable of being expressed by a synthetic gene using methods well known to those skilled in the art, including as described in U.S. Pat. No. 6,506,559 which is incorporated by reference. [0078] a. Pri-miRNA
[0081] The sequence of the pri-miRNA may comprise the sequence of a hairpin referred to in Table 1, the sequence of sequence identifiers 6757248-6894882 of the Sequence Listing of U.S. patent application Ser. No. 10/709,572, the contents of which are incorporated herein by reference, the sequence of sequence identifiers 1-6318 or 18728-18960 of the Sequence Listing of U.S. Provisional Patent Application No. 60/655,094, the contents of which are incorporated herein by reference, or variants thereof. [0082] b. Pre-miRNA
[0084] The sequence of the pre-miRNA may comprise the sequence of a hairpin referred to in Table 1, the sequence of sequence identifiers 6757248-6894882 of the Sequence Listing of U.S. patent application Ser. No. 10/709,572, the contents of which are incorporated herein by reference, the sequence of sequence identifiers 1-6318 or 18728-18960 of the Sequence Listing of U.S. Provisional Patent Application No. 60/655,094, the contents of which are incorporated herein by reference, or variants thereof. [0085] c. MiRNA
[0087] The sequence of the miRNA may comprise the sequence of a miRNA referred to in Table 1, the sequence of sequence identifiers 1-117750 or 6894883-10068177 of the Sequence Listing of U.S. patent application Ser. No. 10/709,572, the contents of which are incorporated herein by reference, the sequence of sequence identifiers 6319-18727 or 18961-19401 of the Sequence Listing of U.S. Provisional Patent Application No. 60/655,094, the contents of which are incorporated herein by reference, or variants thereof. [0088] d. Anti-miRNA
[0090] The sequence of the anti-miRNA may comprise the compliment of a sequence of a miRNA referred to in Table 1, the sequence of sequence identifiers 1-117750 or 6894883-10068177 of the Sequence Listing of U.S. patent application Ser. No. 10/709,572, the contents of which are incorporated herein by reference, the sequence of sequence identifiers 6319-18727 or 18961-19401 of the Sequence Listing of U.S. Provisional Patent Application No. 60/655,094, the contents of which are incorporated herein by reference, or variants thereof. [0091] e. Binding Site of Target
[0095] The present invention also relates to a host cell comprising a vector of the invention. The cell may be a bacterial, fungal, plant, insect or animal cell. 7. Probes
[0112] Hybridization reactions may be accomplished in a variety of ways. Components of the reaction may be added simultaneously, or sequentially, in different orders. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors and anti-microbial agents may also be used as appropriate, depending on the sample preparation methods and purity of the target. [0113] a. Diagnostic
[0115] In situ hybridization of labeled probes to tissue arrays may be performed. When comparing the fingerprints between an individual and a standard, the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes. [0116] b. Drug Screening
[0123] The present invention also relates to a method of using the nucleic acids of the invention as modulators or targets of disease or disorders associated with developmental dysfunctions, such as cancer. In general, the claimed nucleic acid molecules may be used as a modulator of the expression of genes which are at least partially complementary to said nucleic acid. Further, miRNA molecules may act as target for therapeutic screening procedures, e.g. inhibition or activation of miRNA molecules might modulate a cellular differentiation process, e.g. apoptosis. Furthermore, existing miRNA molecules may be used as starting materials for the manufacture of sequence-modified miRNA molecules, in order to modify the target-specificity thereof, e.g. an oncogene, a multidrug-resistance gene or another therapeutic target gene. Further, miRNA molecules can be modified, in order that they are processed and then generated as double-stranded siRNAs which are again directed against therapeutically relevant targets. Furthermore, miRNA molecules may be used for tissue reprogramming procedures, e.g. a differentiated cell line might be transformed by expression of miRNA molecules into a different cell type or a stem cell.
[0124] The present invention also relates to a pharmaceutical composition comprising the nucleic acids of the invention and optionally a pharmaceutically acceptable carrier. The compositions may be used for diagnostic or therapeutic applications. The administration of the pharmaceutical composition may be carried out by known methods, wherein a nucleic acid is introduced into a desired target cell in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation, microinjection, viral methods and cationic liposomes.
[0125] The present invention also relates to kits comprising a nucleic acid of the invention together with any or all of the following: assay reagents, buffers, probes and/or primers, and sterile saline or another pharmaceutically acceptable emulsion and suspension base. In addition, the kits may include instructional materials containing directions (e.g., protocols) for the practice of the methods of this invention.
[0126] We surveyed the entire human genome for potential miRNA coding genes using two computational approaches similar to those described in U.S. Patent Application No. 60/522,459, Ser. Nos. 10/709,577 and 10/709,572, the contents of which are incorporated herein by reference, for predicting miRNAs. Briefly, non-protein coding regions of the entire human genome were scanned for hairpin structures. The predicted hairpins and potential miRNAs were scored by thermodynamic stability, as well as structural and contextual features. The algorithm was calibrated by using miRNAs in the Sanger Database which had been validated.
[0127] Table 2 of U.S. patent application Ser. No. 10/709,572, the contents of which are incorporated herein by reference, shows the sequence ("PRECURSOR SEQUENCE"), sequence identifier ("PRECUR SEQ-ID") and organism of origin ("GAM ORGANISM") for each predicted hairpin from the first computational screen, together with the predicted miRNAs ("GAM NAME"). Table 1 of U.S. patent application Ser. No. 10/709,572, the contents of which are incorporated herein by reference, shows the sequence ("GAM RNA SEQUENCE") and sequence identifier ("GAM SEQ-ID") for each miRNA ("GAM NAME"), along with the organism of origin ("GAM ORGANISM") and Dicer cut location ("GAM POS"). The sequences of the predicted hairpins and miRNA are also set forth on the Sequence Listing of U.S. patent application Ser. No. 10/709,572, the contents of which are incorporated herein by reference.
[0128] Table 1 lists the SEQ ID NO for each predicted hairpin ("HID") of the second computational screen. Table 1 also lists the genomic location for each hairpin ("Hairpin Location"). The format for the genomic location is a concatenation of <chr_id><strand><start position>. For example, 19+135460000 refers chromosome 19, +strand, start position 135460000. Chromosomes 23-25 refer to chromosome X, chromosome Y and mitochondrial DNA. The chromosomal location is based on the hg17 assembly of the human genomc by UCSC, which is based on NCBI Build 35 version 1 and was produced by the International Human Genome Sequencing Consortium.
[0129] Table 1 also lists whether the hairpin is conserved in evolution ("C"). There is an option that there is a paper of the genome version. The hairpins were identified as conserved ("Y") or nonconserved ("N") by using phastCons data. The phastCons data is a measure of evolutionary conservation for each nucleotide in the human genome against the genomes of chimp, mouse, rat, dog, chicken, frog, and zebrafish, based on a phylo-HMM using best-in-genome pair wise alignment for each species based on BlastZ, followed by multiZ alignment of the 8 genomes (Siepel et al, J. Comput. Biol 11, 413-428, 2004 and Schwartz et al., Genome Res. 13, 103-107, 2003). A hairpin is listed as conserved if the average phastCons conservation score over the 7 species in any 15 nucleotide sequence within the hairpin stem is at least 0.9 (Berezikov, E. et al. Phylogenetic Shadowing and Computational Identification of Human microRNA Genes. Cell 120, 21-24, 2005).
[0130] Table 1 also lists the genomic type for each hairpin ("T") as either intergenic ("G"), intron ("I") or exon ("E"). Table 1 also lists the SEQ ID NO ("MID") for each predicted miRNA and miRNA*. Table 1 also lists the prediction score grade for each hairpin ("P") on a scale of 0-1 (1 the hairpin is the most reliable), as described in Hofacker et al., Monatshefte f. Chemie 125: 167-188, 1994. If the grade is zero or null, they are transformed to the lower value of PalGrade that its p-value is <0.05. Table 1 also lists the p-value ("Pval") calculated out of background hairpins for the values of each P scores. As shown in Table, there are few instances where the Pval is >0.05. In each of these cases, the hairpins are highly conserved or they have been validated (F=Y).
[0131] Table 1 also lists whether the miRNAs were validated by expression analysis ("E") (Y=Yes, N=No), as detailed in Table 2. Table 1 also lists whether the miRNAs were validated by sequencing ("S") (Y=Yes, N=No). If there was a difference in sequences between the predicted and sequenced miRNAs, the sequenced sequence is predicted. It should be noted that failure to sequence or detect expression of a miRNA does not necessarily mean that a miRNA does not exist. Such undetected miRNAs may be expressed in tissues other than those tested. In addition, such undetected miRNAs may be expressed in the test tissues, but at a difference stage or under different condition than those of the experimental cells.
[0132] Table 1 also listed whether the miRNAs were shown to be differentially expressed ("D") (Y=Yes, N=No) in at least one disease, as detailed in Table 2). Table 1 also whether the miRNAs were present ("F") (Y=Yes, N=No) in Sanger DB Release 6.0 (April 2005) (http://nar.oupjournals.org/) as being detected in humans or mice or predicted in humans. As discussed above, the miRNAs listed in the Sanger database are a component of the prediction algorithm and a control for the output.
[0133] Table 1 also lists a genetic location cluster ("LC") for those hairpins that are within 5,000 nucleotides of each other. Each miRNA that has the same LC share the same genetic cluster. Table 1 also lists a seed cluster ("SC") to group miRNAs by their seed of 2-7 by an exact match. Each miRNA that has the same SC have the same seed. For a discussion of seed lengths of 5 nucleotides, see Lewis et al., Cell, 120;15-20 (2005).
[0134] The table below shows the information from Table 1 about the miRNA having SEQ ID NO: 8807 and hairpin having SEQ ID NO: 908.
TABLE-US-00001 HID Hairpin Loc C T MID P Pval E S D F LC SC 908 1-204363593 Y G 8807 0.79 0.0013 Y N Y N 42
[0135] The predicted miRNAs from the two computational screens of Example 1 were then used to predict target genes and their binding sites using two computational approaches similar to those described in U.S. Patent Application No. 60/522,459, Ser. Nos. 10/709,577 and 10/709,572, the contents of which are incorporated herein by reference, for predicting miRNAs.
[0137] The table below shows the information from Table 14 about the miRNA having SEQ ID NO: 8807.
TABLE-US-00002 TARGET-GENES ASSOCIATED ROW# DISEASE NAME WITH DISEASE Line Nos. 1 ALL TIMP2, BIRC5, SLC6A3, COMT, 5-178 GIPC2, TSG101, DCC, CDH5, PSMB9, AAT1, TITF1, TGM3, ADAM12, SYN3, CACNA1G, PLP1, AGR2, CES2, CDC2, CYP46A1, ABCD3, ITGA7, RNASE1, HMOX2, SLC3A2, GUCA2A, IPF1, TOP2B, LGALS1, GPR37, F2, UCP2, AQP1, MUC1, SEMA3B, SPRR1B, MAPKAPK3, CBFA2T1, SFRP1, SERPINA1, RRAD, MYLK2, PRKCM PYGM, NUP214, F2RL1, ABCC4, TK2, BMX, TNFRSF5, WFS1, TCL6, HAS2, NEFH, FPGS, CTSK SFN, GUSB, CSH1, HK2, ZIC2, IL6R, MIP, HBA1, DKK3, FASN, MIA, IGF2, SLC6A4, F3, UBC, PCK1, MYBPC3, BLMH, EYA4, CUBN, OGDH, SFTPB, TREM2, ABL1, ATF1, STRC, LUM, ERCC2 GUCA2B, POU3F4, CTAG2, PIM1, P2RY12, TP73L, DDC, DES, DTR IL10, P2RY2, ALDH1A1, UCP3, NDP, RAD52, ITGA6, ADCYAP1, TFAP2C, PCSK1, FXYD3, COL9A3 CD59, MLLT10, TNNT2, MMP2, BMP6, SUCLA2, ABCC3, CRHBP, GPD2, DPYD, LASP1, GALK1, CD33, CHX10, CHRM1, CD24, KCNH1, GPC3, HAS3, IL7R, C4BPB, TMPRSS2, DUT, ALOX12, IL4R, BTC, ATPIF1, DLEC1, CTAG1, DYRK1A, DLEU1, BIRC7, DCT, MGST1, PRM2, C13ORF1, REL, NQO1, MAPK11, HOXB5, SARDH, OR51E2, HOXA6, CTNND1 ZNFN1A2, GAL, NSF, MTAP, FY, CASP6, TAZ, POLK, MT1X, RAD54L, MXI1, MAPK8IP1, LYN, MMP3, MGP, ADRB2, ORC5L, CARD4, HSPA8, S100B, BPGM, EPO, FGL2, PCSK2, ADCY8, BCL2L1, ARHA, TMIE, EGF, PSMB8, SCGB3A1, GNAL, HMGCR, SRD5A2, TCL1A, CD163, CGA, SULT2A1, CD47, ELAC2, ATP1A4 CD8B1, TNFRSF11A, GZMB, MGAT3, UGT1A1, LAT, BCL3, HES1, SERPINI2, TYRP1, PCM1, PRM1, HMOX1, CA12, AVPR2, AMBP, FOSB, DPYSL5, PIGR, HOXA5, HYAL2, BLZF1, ATF3, PBP, CPB2, MBP, PAX2, SP3, AD7C-NTP, SRI, IFNB1, RAGE, RAD51L1, IGFBP4, FBXW7, SEMA3C, UGCG, MSLN, COPEB, TERT, TCL1B, IL3, TNFRSF11B, ATP6V0A4, GNAT2, CD3Z, SIAT9 CCNT1, CD22, EEF2, PTPRF, ELL, BCL2, DSTN, JAG2, GZMA, PRSS8, TMSB10, ATP6V1B1, PTHLH, ALB, APXL, PRODH, IL24, PTPN13, RNF7, ROBO1, RAB3A, THRA, HOXB7, MVK, SLC25A13, IGFALS, MDK, IL6ST MADH1, FLT3LG, CYR61, MBL2, SP1, LAMA1, RASA1, MGAT5, COL6A3, PTH, HDAC1, OPRK1, GRM3, MOG, NFKBIA, PLAB, KCNJ2, RETN, DDIT3, HSPG2, MEST, SCN1A, VIL2, FOSL2, GSTT1, GRIK1, PMS2, PTPRG, COL3A1, VIPR2, AMT, ACVR1, HSD3B2, CREBBP, KRT13, DFFB, CASP5, GSS, RNASEL, SLC26A4, MAPK14, OGG1, MMP10, APOC3, SFXN5, SLPI, NEB, CXCL9, ATP2A1, GNAO1, IL4, CD8A, NBL1, LOX, CCL17, HOXB6, STE CA9, DAPK1, CASR, THRB, HOXA7, CTNND2, FOXA2, IL2RG, AANAT, LAMB1, BRIP1, KCNJ3, IGFBP2, HLF, FRAT1, B2M, ITGA9, LEF1, MAP3K3, IVNS1ABP, SC5DL, LOST1, HMBS TUB, APRT, KLK3, VEGFC, TGM1 VIPR1, FOSL1, HSD3B1, PLD2, GRIK2, CKMT1, DPM3, EIF2S1, ATP10A, TM4SF2, MT3, SNCB, MPZ, IL13, XBP1, TRHR, TRA1, ACP5, APOC2, KRT14, C21ORF107, CD151, GNE, MTCP1 LGALS3, CPLX1, ADAM10, FAP, RASSF1, NONO, MKKS, CNR1, IGFBP1, TPO, GRM5, DAB2IP, LAMB2, PLCL1, ABCC2, STAT5A, GCGR, TNNT1, DIAPH1, THPO, PDGFRL, NDUFV2, CDH6, NFKB2, GADD45G, SYK, CD34, USH1C, TGM2, HLXB9, GPR1, SELP, BMP3, GRB2, GRIK3, NUP88, NISCH, HMGB1, BIRC4, SNCA, NR4A2, F5, CASP3, FUT4, TFPI AXL, CPLX2, ADAM11, RCV1, APEH, TAC1, ARH, PTK2B, PRKCL1, XCL1, MMP1, ITGB7, HOXB8, LCK, JAG1, IGF2R, GABRA5, CARD15, IL1R1, DRD1, ABCC1, G22P1, REN, MPG, CSH2 TFAP2A, STAT5B, NUFIP1, TTR, INHBB, KCNJ11, C3AR1, CRYM, NFKB1, PHKA1, CTSL, JUNB, OGT, LDLR, SCN1B, NID, TIE, ACVR2, HK1, GPR2, IL21R, SCGB1D2, HBA2, GPC1, HAS1, PECAM1, TFG, KLK6, FKBP1B, SFTPC, HOXB3, EDN2, MST1, UBE3A, F2R, NR4A1, SLC9A1, AFM, SSTR1, SNAP25, SLC2A3, RAD51, TYROBP, DSP, UPK3A, IL10RA, CASP8, PIN1, BAG1, FURIN, UCN, TSN, CYP1A1, FUT1, HYAL1, FCGR3A, MLC1, CDC25C, ABAT, CCL21, TSP50, CDC25A, CDK2, BSG, TIEG, CCR2, GOLGA5, ADRA2A, GAD1, GNB3, ARNT, CASP1, FGFR3, CD14, BACE2, TNFRSF10C, FOXN1, ITGAX, MYST3, TULP1, TBX2, ACO1, FUT8, AMACR, LY75, SRPX, POR, ELAVL3, GLDC, TPD52, TBX22, C1QB, CXORF6, CKB, OAS2, MAOB, BIN1, PEA15, NMT1, HBD, PAX4 RECQL, PLA2G2A, BMPR2, CXCL5 PDGFRA, IGSF4, ITGB5, ITGAL, KCNJ6, LIPC, LHX2, MYH2, CDA GRP58, ISGF3G, CALCRL, CCNA1 ICAM2, REA, GABRG2, SERPINB3 HADH2, FBL, SLC4A4, MAP2, RIL, MRPL28, SNCG, ANG, FCGR2A, HLA-DQB1, CDKN2D, CRYAA, NEDD5, IGFBP6, LMNB1, HBB, HSD11B1, CSF2, IL18, HAGH, GPX1, ARHU, PAWR, PGGT1B, SLC16A1, TCAP, DDX10 NP, LEPR, MT1B, GAPD, BAI1, CDH17, AGTR1, CD38, LBP, CXCL13, TGFB2, TIMP4, HSPCB, CNK, IL2RB, BRCA1, ABCA1, TBXA2R, CSK, SLC6A1, KLK4, PCMT1, GATA2, RTN1, GAP43, SST, COPS5, SLC2A5, CXCR4, SELE, AQP2, NKX3-1, MOBP, ITGA5, EPHA4, DLST, COCH, CST3, TSC1, UCHL1, IFNG, SI, SULT1A1, HTR2A, PRF1, RSN, ENTPD1, PRKR, FYN, GPT, HTR1D, DRD4, MSF, AXIN1, GHRL, LTF, PDE4D, F8, SELL, ARHC, MAZ, INSR, KRT18, CHUK ENPP2, KCNA5, ITGB3, BMP1, MEF2A, ADAM2, PRDX1, CDK9, APOL4, TGFBI, GABRA1, MALT1, TPT1, CD1D, CUL2, EPHB2, ITGA2B, TCF7, RORA, NR1I2, HRASLS, TGFBR2, CSPG2, COL1A2, THBS2, ZNFN1A1, DPH2L1, TU3A, KRTHB1, KIT, SKP2, TTN, ALOX5AP, LYZ, PRKCA, SLC18A2, CTSH, CUTL1, PRY, HTR3A, MSH2, TNFSF12, BAD, PON1, NRP1, DTNBP1, FABP4, MYO1A, PRLR, IL16, MC1R, EXTL3, RBP1L1, TPM2, SCA7, ADAMTS4, AKR1B1, MADH4 RALA, DAF, DAB2, TAT, TNF, CCS, ACP1, SIM1, CCL7, TK1, ALOX5, RDS, NCOA2, BUB1, ITGA3, FPRL1, SLC26A3, MAX, MX1, ACY1, CHRNB2, EIF4A2, XRCC5, RRAS, CCND2, COL4A4, ADPRT, CANX, FHIT, GCK, FOXP3, LILRB1, ILK, SNRPB, CR1, ATM, ATP2A2, CBS, CCNC, EPHA2, TPI1, MAP3K4, FGF5, FADS1, BCL10, CDH1, FTH1, NOS3, PURA, ZXDA, TXN, SYCP1 SHH, GSR, PRKCQ, ASCL1, CYP11A1, SLC5A7, SDC2, PGF, SOD2, NR3C2, CST, DRD2, C6ORF18, OSMR, SFTPA2, RANBP17, RPGR, SYBL1, HSD17B3, GCSH, BCHE, PLD1, PLAU, APBA1, YES1, ABCC8, ANXA2, ORM2, MYL3, ABP1, GABRA3, STK11, PTCRA, APP, BLM, CRH, DBH, TNFRSF8, EPS15, DDR1, SMARCB1, VIM, LRPAP1, DAO, GGH, MAPT, MAP1B, HGFAC, PPP2CB, MYO18B GALT, HOXA11, TJP2, E2F4, WNT10B, BCL11A, GPI, FABP2, F12, MLF1, ACTA1, MYO3A, DCN HSPA5, TCTA, OPHN1, CFLAR, NCL, TRAPPC1, GFAP, SCGB3A2, MLN51, MUC4, LRP5, CCL5, RPA1, MNT, ATP1A1, BRAF, BLR1, PPP2R1B, MATK, ENO2, WNT7A, PARK2, S100A9, IMP-2, TNFSF10, COL4A2, RDX, LMO1, VSNL1, CFTR, IGF1, GABRB3, UBB, CHST2, ACVR1B, SERPINF2 ITM2B, DIA1, EYA1, PTK6, MTRR, ADM, UMOD, CACNA2D2, KLF5, CEBPA, FPR1, TFE3, ABCD1, CDK10, RBBP1, BIRC3, XRCC3, WHSC1, TALDO1, DIO2, OPRM1, PRKCZ, CAB2, CBL, SLC5A5, LIG4, SELPLG, FCGR1A FRK, COL6A1, DNMT3B, ALDOA, MS4A2, CRKL, APOL2, FIGF, IDS, NTRK3, ASGR1, HDGF, RAG2, SMN1, PADI4, GLUL, ALAS2, APOC1, VDR, SSB, PPARA, GNAQ, HMGA2, HOXA1, HOXA13, ME1, TNFSF7, LIPE, PLCD1, SERPINE2, EREG, KLK13 VLDLR, TFF3, TBXAS1, LYL1, APOBEC1, KCNJ8, CALCB, PRND, KLKB1, MMP12, EGR1, MLLT7, LMNA, MAP4, SRF, BUB1B, CHI3L1, C4A, ATP7B, LTA, MYCL1, CD97, UMPK, RNF14, TG SOCS3, GSN, SIL, PRDM2, NOTCH1, BCL6, EVPL, PNOC, GAS6, SLC1A2, ANPEP, DSCAM, VIL1, KCNQ2, GHRHR, GRIN1, YWHAG, MMP8, XK, ACHE, DYT1, C5, ELAVL2, TYMS, IL7, EIF3S3, NEK4, ACTG1, PITX3, CDSN, MAOA, NUP98, MDS1, PFKM, FES, CDC27, CXADR, SERPINI1, NRP2, COL17A1, DTYMK, PAX3, PCSK7, VIP, SYP
FN1, ITGAM, CAPN10, PTPRC, AARS, DDX6, HSPD1, CYP21A2, CSF3, AK1, MADH5, ED1, CSRP3 ENC1, MTP, IGFBP7, TPM1, VHL FLT1, GRM7, NCOA1, MMP9, CETP, MME, PRTN3, TYRO3, LMO2, APRIN, HP, FKBP1A, ETS2, SFTPD, COL4A1, GRIK5, EBAG9, TGIF, SIAT8A, RUNX1, SLC2A4, ANGPT4, NRAS, AF3P21 EDN1, HSPCA, CPA5, REG1A, CHEK2, ECE1, NPPA, CASP9, MAPK10, BDNF, DNASE1, CEBPB, FRZB, HPN, CYP1A2, SERPINH1, MPL, KLF4, ADORA1, SLC11A1, CSTB, DOK1, PDPK1, GRB7, GAD2, CDC25B, APOH, EPAS1, ING1, CCR3, CD68, ESRRA, FVT1, AF5Q31, APCS, UBE2I, FGF8, WNT3A, HSF4, HNMT, MAPKAPK2, PAM, FBN2, RYR2, DPP4, STAT6, KRT17, FLJ11383 MFGE8, CRHR1, MS4A1, NTF5, APOA2, AKT1, CIRH1A, ACTC, TDGF1, CYP19A1, LAMR1, GNAI2 ADAMTS13, JUN, MBD4, POLB, MT2A, DNTT, NROB2, EEF1A2, VTN, FUT7, ARVCF, DUSP1, ANXA3, ABCG2, SSX2, TPM3, CCKAR, MEIS1, MSN, BAALC, EBNA1BP2, PTGS1, IGF1R, ACTN4, COL1A1, ASPSCR1, PKLR NUP153, FGG, SERPING1, DSCR8 TGFBR3, PHF11, STS, EXO1, CX3CR1, GABRG3, PON2, CBFA2T3, MFI2, CLDN14, CYP11B2, MAT1A, EPB41L3, PIP IGFBP5, NEUROD1, SYN2, PTPN1 PRKCG, PAPPA, PEPD, MEFV, PTGER4, MADH3, ELAVL4, TNFSF11, ENPP1, STC1, MYBL2, LRP2, SIM2, USP4, CAST, ARMET, EIF4A1, AGTR2, DCTD, ITGA4, ALPL, RABEP1, CTPS, WNT8B, CDX2, DSCR1, WNT1, SLC6A2, VCY2, DSG1, CCND1, HOXB4, CCKBR, CXCL12, ERBB4, RAD50, FZD10, GATA1, B3GALT2 FRDA, CCK, CD4, ACPP, CASP7, DEXI, HCK, FADS2, PROS1, LPL SERPINF1, EPHA3, CTSW, BACE, HSD17B2, SLC6A14, HSD3B7, ASC, GPRK5, PCBD, CCR1, PBX1 MAT2A, CDK5R1, CDC10, ZAP70, PGK1, ABCC5, DRD5, ATRX, JUP APBA2, MYL4, F9, BID, RELN, WAS, XPC, SLC19A2, MYL2, TNFRSF9, TERF1, PPP1CC, KCNJ9, ANXA1, ENO1, WT1, HMGA1, ERCC5, DBI, CHRNA3, IL15, BDKRB2, MATN3, CHK, NGFB, MAPK1, SH2D1A, CART, CAT, ALCAM, SIAH1, MBD2, GNA11, NR2F6, CSE1L, ETV4, DDB1, CHRNE, CLU, LTB, SLC4A1, APM1, CTSG, FABP3, MYO6, PLK, IRF4, EGFR, BAX, SRP19, STAT1, BCL9, TH, PACE4, SPG7, MYOD1, CCL4, ATP1A2, DAB1, ABCB11, LTC4S, L1CAM, HRG, ORM1, PDGFB, PRG2, THY1, CAV1, SERPINB5, F11, CHRNB1, CD37, PKD1, CCND3, RXRG, PREP, HRAS, UPP1, GARP, ALOX15, ITGA2, SLC22A4, CXCR3, CXCL10, NGFR NCAM1, WWOX, NME2, CNN1, NMB TOP1, A2M, ZNF151, SLC2A10, MSMB, TIA1, BIRC2, HOXC11, RB1, AGER, SOD3, RECK, ANP32A, RBP1, CD3G, CLCN5, P2RX7, NEF3, ARX, PLA2G10, NR3C1, AR, BAK1, RB1CC1, DRD3, DBP, SFTPA1, SEL1L, NTRK2, F7, DISCI, AMFR, MAPK8, TIAM1, NEUROG3, AXIN2 PROM1, RHD, MRE11A, AVPR1A, MYO7A, TCF1, USF1, VCL, TLX1 SERPINA5, RGS4, PFN1, SMS, SCP2, SPINK1, LCN2, CHRNA1, FOLR2, C1QA, GABBR1, UBTF, HADHA, NAP1, C7, TFF2, FOS, IL5, PLTP, OXT, PICALM, CASP2, ALS2, SPN, C1S, RFC3, PAX5, TPX2, SNAI1, TF, DIRC2 FDXR, MYO15A, CYLD, MMP13, SEMA3F, LMOD1, S100A1, FABP1 INSM1, TJP1, MHC2TA, HOXB9, HLA-DRB3, TRAF1, PCAF, WHSC1L1, TPK1, HR, GPX4, CD9 ACE, HSD11B2, SLC1A1, IRF2, TNFSF6, STMN1, DYSF, ADAM17, TMPRSS3, PXN, DCX, PRKAG2, KCNQ1, ABCA4, SIAT6, IMP-1, GRPR, SLC2A2, PLU-1, HLA-DMB KRAS2, NSEP1, AZGP1, NLGN4, IL12A, SEPP1, MAD, CASQ2, USH3A, GSTP1, CR2, MJD, COL4A3, PTPRJ, WHSC2, MTM1, GUCY2C, CEACAM8, PDCD8, CST6 LZTS1, HARS, CRABP1, MAP2K1, PRKAA2, EPM2A, HRH1, SORD, NR5A1, NPR3, CTNNBIP1, HOXA9 TIF1, TNNI3, PLAT, LEP, HBZ, ITGA11, SDC1, LAMB3, NPY5R, SOD1, ADD1, TAL1, COL6A2, MAPK6, SREBF1, POLG, SLC7A5, MDM2, CD44, FGFR4, GJB6, CEACAM1, NTF3, NF1, APOA4, CPE, APOL1, HMMR, YY1, PNMT, COMP, CCL1, S100A7, UBE2B, ALDH3A1, MAP3K8, FGF1, DBN1, SCGB1A1, P8, GSTA2, CPT2, CXCR6, NR2F2, MTNR1A, ARHI, ARG2, SPINT2, MMP15, RXRB, SCN5A, CTSD, BCAR1, COX4I1, XRCC1, IAPP, CTSS, NUMA1, DLD, CHRNA7, BCAS2, CRYGD, GALNT3, GLI, CYP27A1, MTHFR, CALCA, MYH9, NCF2, CRYGS, CHGB, PTPRA, PTAFR, CLDN1, COL11A2, AKR1B10, DCK, JAK3, TCF4, CS, CTNNA1, SREBF2, COL18A1, HIP1, HTR2C, FBLN5, RRAS2, APEX1, SRC, FBP1, FKBP4, MLLT2, FABP7, MDM1, SLC1A3, PDYN, HSPB1, NPY, PLEC1, CRYAB, FANCA, UPK2, THBS4, EDNRA, KCNN3, LRP1, EPOR, DSC3, ERBB2, IL2RA, CLOCK, GSTA1, S100A6, CCL2, PDE5A, NR2F1, CALB2, GFRA1, CYP7A1, DSCR3, ST14, KRT8, PSEN1, SCN4A, CNP, LGI1, KITLG, CX3CL1, XRCC2, STARD3 PGC, ADRBK1, CYP1B1, CRYGC, GATA3, HNRPA1, KL, CYP2D6, NCSTN, NPHP1, DAZL, KIAA1199 MAP2K6, ZNF145, CHAT, EIF4E, FADD, TACC3, FACL4, UFD1L, PTGES, IL1RL1, FOLH1, FKBP5, CLDN11, ZNF198, WRN, GPR30, IRF1, CDH23, RET, MLLT1, JRK IL13RA2, TFCP2, FGFR2, LAMP3 PLCG1, ID1, CSN2, PCSK9, HF1 KLF1, MAG, FHL2, NRIP1, SAH, CEACAM5, MKL1, CTSB, FAAH, ERBB3, AMN, GCNT2, CYP7B1, NCOR1, WISP1, ADCY1, MECP2, GSTM1, SPARC, CALB1, CREB1, COL4A6, CHRNA5, KLK10, PSEN2 MYH7, UPK1A, MAPK3, IRTA1, CDX1, GADD45B, BCAR3, NRG1, FSHR, RBL2, CCNB1, INPPL1, DEK, GJB1, ADRA2C, AZU1, HSD17B1, PHB, COL2A1, MAP2K5 DLGAP2, GRIA1, CACNB2, JAK1, BRS3, SLC18A3, SCG2, IRS1, FABP5, FLT3, BZRP, FST, PTTG1, NCOA3, GNMT, CD74, CD83, TSLP, PKM2, HOXC5, GCKR, CA1, ACTB, IL1RN, SLC19A1, LAMP2, PAH, MSR1, PTN, AVP, FGFR1, HOXD4, ENO3 PPID, GPR44, SCYE1, NR0B1, DP1, EMS1, ADCY2, TTC3, ERG, FCAR, CDKN2C, CHRNA4, AGT, DLAT, UPK1B, RPL19, C5ORF7, DIABLO, MLN, AURKB, KAI1, NCF1, PITPNM1, TP53, CHGA, XLKD1, NDRG1, FLJ22795, PTPRN, CYP27B1, JAK2, TFRC, HOXD3, MITF, IRS2, BMPR1A, CRAT, C11ORF17, CTNNB1, GJB2 PTOV1, CORTBP2, HTR2B, SAT, PRNP, LDHA, MAP2K4, FLT4, MMP16, GRIA2, CREM, MLLT3, DNMT1, MST1R, ST7, AIRE, GCLC, AIF1, ADA, RAN, MTA1, AFP, PRKAR1A, RPS6KA1, HOXC4 SSA2, APBB1, NLGN3, TRPV6, NOV, CYP3A4, WNT2, MEP1A, NR2C1, LAMP1, MSX1, MPO, NBS1, PTGIS, CDKN2B, OMG, RPL29, GFPT1, ITGB2, LTB4R, PPP1R3A, PIAS3, NCAM2, PPM1D NOS2A, HPSE, CDK6, TTC4, HOXA10, PRKDC, PPARD, ELF3, VCAM1, ALK, CSEN, PPARGC1, APLP2, PTMA, SERPINE1, PRSS1 TP73, TMEFF2, GRIA3, CACNB4, BCL11B, CTGF, PBOV1, CXCL1, CRABP2, TACR1, SPDEF, TNC, CDH11, FGA, SCD, GLP1R, HBG1 HNF4A, C9, WISP2, HSF1, CD63 C21ORF33, EPHX1, GSTM3, KCNQ3, PLCB1, CCR6, POMC, GRN, SSA1, MET, CDC42, ACR, ADSL, SOX13, RPS6KB1, PTGER2 MCC, MAF, WNT3, ELOVL4, CRYBB1, YWHAH, HRH3, CDKN1B, LCP1, BSND, TGFB3, RPS6, LIM2, CD19, CDKN2A, NCOR2, ITGB1, CEACAM6, GNAS, OPRD1, RARG, MAP2K3, DMD, CDK7, MYH6, INA, EFNA1, MAD2L1, ENPEP, TEK, TIMM8A, SS18, GADD45A, PRCC, APLP1, TKT, STAT2, PTPRN2, CXCL2, SGCD, FGB, PAX7, GPA33, ICA1, SCG3 PRSS2, PTHR1, SCT, G6PD, EMP1, IDE, HBG2, OCLN, CP, CTLA4, CCR7, LUC7L, NCOA4, GLI2, THBD, RAP1A, CAV2, CA2 GP9, TRAF4, SMAP1, PRKCD, GRP, CHL1, KCNN4, SLC2A1, CDKN1A, SQSTM1, GFRA2, TCP1, SMO, MADH6, CCL3, CCNI, NOS1 SOAT1, RBM5, DDX26, EZH2, COX15, RPN1, CALCR, NME1, ANGPT1, CDK4, ECGF1, CALR, CCR4, MTR, PAK1, SOX4, PIK3R1, IL1B, AKT2, SCA2, GAS, MYCN, GHR, KRT7, PCNA, TMC1, FBN1, ERN1, CDH13, SLC25A1, HFE, BGLAP, F13A1, IL8RA, MEN1, MAGEC1, G6PC, SHC1, ATP6V1G2, ATIC, SNRPN, HNRPA2B1, FMR1, IL13RA1, USF2, TCF2, APPBP2, PFKL, OTOF, CRMP1, TNNC1, PTPN6, LCN1, BARD1, AREG, MADH7, DHCR24, TFAM, TAGLN, ENG, DFNA5, LPP, NPY2R, EFNB2, ANGPT2, VWF, ALPP, MBD1, BCAS1, RBM6, DSCR5, MSH6, ADORA3, ICAM3, CDK5, SLC22A1L, RPN2, SPINT1, BLCAP, IL1A, AKT3, AOC3, TACC1, AHR, ASAH1, GYS1, GC, MYF6, SET, RELA, HD, MLH1,
TNFRSF1B, CNTF, PRL, SAA1, GRIN2B, FMR2, CCL20, GOLGB1, IL8RB, CCR5 and LAMC2. 24 HIV ABCC5, ALOX5, ALOX5AP, 277-289 ANP32A, APM1, AR, BCL2, BCL2L1, BDKRB2, BGLAP, BLR1, BSG, C5, CASP3, CASP8, CAT, CAV1, CCL1, CCL2, CCL4, CCL5 CCNT1, CCR2, CCR3, CCR4, CCR5, CCR7, CD14, CD22, CD34 CD38, CD3G, CD4, CD44, CD59, CD63, CD68, CD8A, CD8B1, CDC2, CDKN1A, CR1, CSF3, CTLA4, CTSD, CX3CL1, CX3CR1, CXCL10, CXCL12, CXCR4, CXCR6 CYP3A4, DPP4, EPO, FCAR, FKBP1A, FOS, FPR1, FURIN, FYN, GOLGB1, GPR1, GPR44, GZMA, GZMB, HARS, HCK, HLA- DRB3, HNRPA1, HNRPA2B1, HP, ICAM2, ICAM3, IL10, IL12A, IL13, IL15, IL16, IL18, IL1A IL1RN, IL2RA, IL2RB, IL6R, IL6ST, IL7, IL8RB, ITGAL, ITGAX, ITGB3, JUNB, KIT, KITLG, LCK, LEP, LILRB1, LTA LTB4R, LTF, MAPK3, MBL2, MHC2TA, MSN, NCAM1, NCL, NGFR, NMT1, NPY, NR3C1, NUP214, OCLN, OPRD1, OPRK1, PACE4, PCSK1, PLAT, PLAU, PLEC1, POMC, PRF1, PRKR, PTPRC, PURA, PXN, RELA, SELE SELL, SELP, SLC2A1, SLC2A3, SLPI, SPN, SREBF1, STAT1, STAT5A, TAC1, TACR1, TFRC, TG, THBD, TIA1, TJP1, TNFRSF1B, TNFRSF5, TNFRSF8, TNFSF10, TNFSF6, TNFSF7, TP53, TXN, VIPR1, VWF, XCL1 AND ZAP70 27 Lymphoma ADAM12, ALOX5, ATIC, ATRX, 296-304 BCL10, BCL11A, BCL2, BCL3, BCL6, BCL9, BLMH, BLR1, BUB1 CALR, CBL, CCND1, CCND3, CCR4, CCR6, CCR7, CD24, CD37 CD97, CDC10, CDC25A, CDC25B, CDK10, CDKN2C, CFLAR, COL18A1, CTAG1, CXCL12, DAF, DAPK1, DDX6, EIF4A2, ENTPD1, EZH2, FBLN5, FCGR1A, FES, FLT4, FVT1, FYN, HOXC5, HSPCA, ICAM3, IL10, IL16, IL21R, IL4R, IL6R, IRTA1, ITGA5, ITGB7, JAK3, JUNB, LCP1, LEF1, MAD2L1, MALT1, MHC2TA, MLLT10, MMP9, MPZ, MRE11A, NBS1, NP, PAX5, PCSK7, PIAS3, PIM1, PLK, PRDM2, PTPN6, RAD54L, RBL2, SDC1, SERPINB5, SH2D1A, SKP2 SOCS3, SRC, SRI, SSX2, STAT1 TAL1, TCF7, TCL1A, TCL1B, TFG, TNF, TNFRSF5, TNFSF7, TP53, TP73, TPM3, TRAF1, ZNF198 AND ZNFN1A1 38 Leukemia ABCG2, ABL1, ADAM12, AF3P21, 375-391 ALOX5, ANG, ARNT, AXL, BAALC BCL10, BCL11A, BCL11B, BCL3, BCL9, BIRC4, BLR1, BLZF1, BUB1, C13ORF1, C5ORF7, CACNA1G, CASP2, CASP3, CAST, CBFA2T1, CBFA2T3, CBL, CCNC, CCND3, CCR6, CCR7, CD163, CD24, CD37, CD44, CD47, CD8A CDA, CDH17, CDKN2C, CDKN2D, CEACAM6, CEBPA, CFLAR, CKB, COL18A1, CREBBP, CRKL, CSE1L CSPG2, CTGF, CTPS, CTSW, CUTL1, CXCL12, CXCR3, CYP3A4 DAF, DAPK1, DCK, DDX10, DEK, DLEU1, DNMT3B, DOK1, ELL, ENTPD1, EPOR, EPS15, ERG, EZH2, F11, FADS2, FES, FGF1, FGFR3, FLT4, FOLR2, FPGS, FYN, G22P1, GADD45A, GADD45G GATA2, GGH, GPR37, GRB2, HCK HDAC1, HIP1, HNRPA1, HOXA10, HOXA11, HOXA13, HOXA6, HOXA7 HOXA9, HOXB4, HOXB5, HOXB6, HOXB7, IL2RB, IL3, ISGF3G, ITGA5, ITGB7, JAK2, JAK3, JUNB, KIT, LCK, LHX2, LMO1, LMO2, LPP, LTC4S, LYL1, MAD2L1, MEIS1, MKL1, MLLT1, MLLT10, MLLT2, MLLT3, MLLT7, MSF, MTAP, MTCP1, MYST3, NBS1, NCL, NCOA2, NCOR2, NEDD5, NOTCH1, NP, NQO1, NTRK3, NUMA1, NUP214, NUP98, OAS2, ORC5L, P2RX7, PBX1, PDGFRA, PECAM1, PHKA1, PICALM, PRDX1, PRKDC, PRKR, PTPN6, PURA, RABEP1, RANBP17 RPN1, SELL, SELPLG, SERPINF2 SET, SH2D1A, SIL, SKP2, SLC19A1, SLC9A1, SOCS3, SRC, STAT1, STAT2, SYK, TAL1, TCL1A, TCL6, TCTA, TERF1, THY1, TIA1, TIE, TLX1, TM4SF2, TNF, TNFSF7, TP73, TRAF1, TSN, TXN, VDR, WHSC1L1, XRCC5, ZNF145, ZNFN1A1 AND ZNFN1A2. 122 Medullary CALCB, GFRA1, GFRA2, NTRK3 574-575 thyroid AND RET. carcinoma
[0138] Table 4 lists the predicted target gene for each miRNA (MID) and its hairpin (HID) from the second computational screen. The names of the target genes were taken from NCBI Reference Sequence release 9 (http://www.ncbi.nlm.nih.gov; Pruitt et al., Nucleic Acids Res, 33(1):D501-D504, 2005; Pruitt et al., Trends Genet., 16(1):44-47, 2000; and Tatusova et al., Bioinformatics, 15(7-8):536-43, 1999). Target genes were identified by having a perfect complimentary match of a 7 nucleotide miRNA seed (positions 2-8) and an A on the UTR (total=8 nucleotides). For a discussion on identifying target genes, see Lewis et al., Cell, 120: 15-20, (2005). For a discussion of the seed being sufficient for binding of a miRNA to a UTR, see Lim Lau et al., (Nature 2005) and Brenneck et al, (PLoS Biol 2005).
[0140] The table below shows the information from Table 4 about the miRNA having SEQ ID NO: 8807 and hairpin having SEQ ID NO: 908.
TABLE-US-00003 HID MID Target Genes and Binding Sites 908 8807 ATP6V1A (120264); BCL9 (120255); C1QTNF3 (120265); C20orf85 (120276) CACNG4 (120275); CAPN3 (120273); CD8B1 (120263); CHRFAM7A (120274) CHRNA7 (120272); CTPS (120254); DPYSL3 (120266); EPHA7 (120269); GFRA (120270); HEBP2 (120268); MEIS1 (120262); NFASC (120259); PREL (120258); RABIF (120261); S100PBPR (120253); SNX27 (120257); SURF (120271); TBN (120267); TCEA3 (120260); TSRC1 (120256)
[0142] The table below shows the information from Table 5 about the miRNA having SEQ ID NO: 8807.
TABLE-US-00004 HID MID Dis N T Per. Pval Target Gene Names 908 8807 116 1 7 14.3 0.0117 GFRA2 908 8807 109 3 269 1.1 0.0101 BCL9, CTPS, MEIS1
[0143] The table below shows the information from Table 8 about the miRNA having SEQ ID NO: 8807.
TABLE-US-00005 Disease Name ID Leukemia 109 Medullary thyroid carcinoma 116
[0144] Table 6 shows the relationship between the miRNAs ("MID")/hairpins ("HID") and diseases by their host genes. We defined hairpins genes on the complementary strand of a host gene as located on the gene: Intron_c as Interon and Exon_c as Exon. We choose the complementary strands as they can cause disease. For example, a mutation in the miRNA that is located on the complementary strand. In those case that a miRNA in on both strands, two statuses like when Intron and Exon_c Intron is the one chosen. The logic of choosing is Intron>Exon>Intron_c>Exon_c>Intergenic. Table 9 shows the relationship between the target sequences ("Gene Name") and disease ("Disease Code").
[0145] The table below shows the information from Table 9 about the miRNA having SEQ ID NO: 8807.
TABLE-US-00006 Gene Name Disease Code BCL9 109, 112 CD8B1 92 CHRNA7 8, 24, 144, 162 CTPS 109 MEIS1 109 GFRA2 116
[0146] Validation of miRNAs
[0147] To confirm the hairpins and miRNAs predicted in Example 1, we detected expression in various tissues using the high-throughput microarrays similar to those described in U.S. Patent Application No. 60/522,459, Ser. Nos. 10/709,577 and 10/709,572, the contents of which are incorporated herein by reference. For each predicted precursor miRNA, mature miRNAs derived from both stems of the hairpin were tested.
[0148] Table 2 shows the hairpins ("HID") of the second prediction set that were validated by detecting expression of related miRNAs ("MID"), as well as a code for the tissue ("Tissue") that expression was detected. The tissue and diseases codes for Table 2 are listed in Table 7. Some of the tested tissues wee cell line. Lung carcinoma cell line (H1299) with/without P53: H1299 has a mutated P53. The cell line was transfected with a construct with P53 that is temperature sensitive (active at 32° C.). The experiment was conducted at 32° C.
[0149] Table 2 also shows the chip expression score grade (range of 500-65000). A threshold of 500 was used to eliminate non-significant signals and the score was normalized by MirChip probe signals from different experiments. Variations in the intensities of fluorescence material between experiments may be due to variability in RNA preparation or labeling efficiency. We normalized based on the assumption that the total amount of miRNAs in each sample is relatively constant. First we subtracted the background signal from the raw signal of each probe, where the background signal is defined as 400. Next, we divided each miRNA probe signal by the average signal of all miRNAs, multiplied the result by 10000 and added back the background signal of 400. Thus, by definition, the sum of all miRNA probe signals in each experiment is 10400.
[0150] Table 2 also shows a statistical analysis of the normalized signal ("Spval") calculated on the normalized score. For each miRNA, we used a relevant control group out of the full predicted miRNA list. Each miRNA has an internal control of probes with mismatches. The relevant control group contained probes with similar C and G percentage (abs diff <5%) in order to have similar Tm. The probe signal P value is the ratio over the relevant control group probes with the same or higher signals. The results are p-value <0.05 and score is above 500. In those cases that the SPVa1 is listed as 0.0, the value is less than 0.0001.
[0151] The table below shows the information from Table 2 about the miRNA having SEQ ID NO: 8807 and hairpin having SEQ ID NO: 908.
TABLE-US-00007 HID MID Tissue S SPval Disease R RPval 908 8807 10 17708 0.0007 908 8807 3 14850 0.0033 908 8807 4 4099 0.0113 908 8807 11 88104 0 908 8807 6 4537 0.0033 908 8807 7 16554 0.0013 908 8807 7 2193 0.0127 908 8807 9 107208 0 908 8807 13 2941 0.004 908 8807 14 22436 0.0027 908 8807 12 20341 0.0007 908 8807 5 6600 0.0033 908 8807 1 6.05 0.0016 908 8807 2 0.69 0.0076
[0152] The following table shows the information from Table 7 about the miRNA having SEQ ID NO: 8807 and hairpin having SEQ ID NO: 908.
TABLE-US-00008 Tissue or Disease name ID Prostate adenocarcinoma 1 Lung adenocarcinoma 2 Skeletal muscle 3 Spleen 4 Lung 5 Lung adenocarcinoma 6 Placenta 7 Prostate adenocarcinoma 9 Prostate 10 Brain Substantia Nigra 11 Testis 12 Uterus carcinoma cell line (HeLa) 13 Adipose 14
[0153] To further confirm the hairpins and miRNAs predicted in 0, we detected expression in additional tissues. Table 2 of U.S. Provisional Patent Application No. 60/655,094, which is incorporated herein by reference, lists expression data of miRNAs by the following: HID: hairpin sequence identifier for sequence set forth in the Sequence Listing of U.S. Provisional Patent Application No. 60/655,094, which is incorporated herein by reference; MID: miRNA sequence identifier for sequence set forth in the Sequence Listing of U.S. Provisional Patent Application No. 60/655,094, which is incorporated herein by reference; Tissue: tested tissue; S: chip expression score grade (range=100-65000); Dis. Diff. Exp.: disease related differential expression and the tissue it was tested in; R: ratio of disease related expression (range=0.01-99.99); and abbreviations: Brain Mix A--a mixture of brain tissue that are affected in Alzheimer; Brain Mix B--a mixture of all brain tissues; and Brain SN--Substantia Nigra.
[0154] To further validate the hairpins ("HID") of the second prediction, a number of miRNAs were validated by sequencing methods similar to those described in U.S. Patent Application No. 60/522,459, Ser. Nos. 10/709,577 and 10/709,572, the contents of which are incorporated herein by reference. Table 3 shows the hairpins ("HID") that were validated by sequencing a miRNA (MID) in the indicated tissue ("Tissue").
[0155] A group of the validated miRNAs from Example 3 were highly expressed in placenta, have distinct sequence similarity, and are located in the same locus on chromosome 19 (FIG. 2). These predicted miRNAs are spread along a region of ˜100,000 nucleotides in the 19q13.42 locus. This genomic region is devoid of protein-coding genes and seems to be intergenic. Further analysis of the genomic sequence, including a thorough examination of the output of our prediction algorithm, revealed many more putative related miRNAs, and located mir-371, mir-372, and mir-373 approximately 25,000bp downstream to this region. Overall, 54 putative miRNA precursors were identified in this region. The miRNA precursors can be divided into four distinct types of related sequences (FIG. 2). About 75% of the miRNAs in the cluster are highly related and were labeled as type A. Three other miRNA types, types B, C and D, are composed of 4, 2, and 2 precursors, respectively. An additional 3 putative miRNA precursors (S1 to S3) have unrelated sequences. Interestingly, all miRNA precursors are in the same orientation as the neighboring mir-371, mir-372, and mir-373 miRNA precursors.
[0156] Further sequence analysis revealed that the majority of the A-type miRNAs are embedded in a ˜600bp region that is repeated 35 times in the cluster. The repeated sequence does not appear in other regions of the genome and is conserved only in primates. The repeating unit is almost always bounded by upstream and downstream Alu repeats. This is in sharp contrast to the MC14-1 cluster which is extremely poor in Alu repeats.
[0157] FIG. 3-A shows a comparison of sequences of the 35 repeat units containing the A-type miRNA precursors in human. The comparison identified two regions exhibiting the highest sequence similarity. One region includes the A-type miRNA, located in the 3' region of the repeat. The second region is located ˜100 nucleotides upstream to the A-type miRNA precursors. However, the second region does not show high similarity among the chimp repeat units while the region containing the A-type miRNA precursors does (FIG. 3-B).
[0158] Examination of the region containing the A-type repeats showed that the 5' region of the miRNAs encoded by the 5' stem of the precursors (5 p miRNAs) seem to be more variable than other regions of the mature miRNAs. This is matched by variability in the 3' region of the mature miRNAs derived from the 3' stems (3 p miRNAs). As expected, the loop region is highly variable. The same phenomenon can also be observed in the multiple sequence alignment of all 43 A-type miRNAs (FIG. 4).
[0159] The multiple sequence alignment presented in FIG. 4 revealed the following findings with regards to the predicted mature miRNAs. The 5 p miRNAs can be divided into 3 blocks. Nucleotides 1 to 6 are C/T rich, relatively variable, and are marked in most miRNAs by a CTC motif in nucleotides 3 to 5. Nucleotides 7 to 15 are A/G rich and apart from nucleotides 7 and 8 are shared among most of the miRNAs. Nucleotides 16 to 23 are C/T rich and are, again, conserved among the members. The predicted 3 p miRNAs, in general, show a higher conservation among the family members. Most start with an AAA motif, but a few have a different 5' sequence that may be critical in their target recognition. Nucleotides 8 to 15 are C/T rich and show high conservation. The last 7 nucleotides are somewhat less conserved but include a GAG motif in nucleotides 17 to 19 that is common to most members.
[0160] Analysis of the 5' region of the repeated units identified potential hairpins. However, in most repeating units these hairpins were not preserved and efforts to clone miRNAs from the highest scoring hairpins failed. There are 8 A-type precursors that are not found within a long repeating unit. Sequences surrounding these precursors show no similarity to the A-type repeating units or to any other genomic sequence. For 5 of these A-type precursors there are Alu repeats located significantly closer downstream to the A-type sequence.
[0161] The other miRNA types in the cluster showed the following characteristics. The four B group miRNAs are found in a repeated region of ˜500bp, one of which is located at the end of the cluster. The two D-type miRNAs, which are ˜2000 nucleotides from each other, are located at the beginning of the cluster and are included in a duplicated region of 1220 nucleotides. Interestingly, the two D-type precursors are identical. Two of the three miRNAs of unrelated sequence, S1 and S2, are located just after the two D-type miRNAs, and the third is located between A34 and A35. In general, the entire ˜100,000 nucleotide region containing the cluster is covered with repeating elements. This includes the miRNA-containing repeating units that are specific to this region and the genome wide repeat elements that are spread in the cluster in large numbers.
[0162] To further validate the predicted miRNAs, a number of the miRNAs described in Example 4 were cloned using methods similar to those described in U.S. patent application No. 60/522,459, Ser. Nos. 10/709,577 and 10/709,572, the contents of which are incorporated herein by reference. Briefly, a specific capture oligonucleotide was designed for each of the predicted miRNAs. The oligonucleotide was used to capture, clone, and sequence the specific miRNA from a placenta-derived library enriched for small RNAs.
[0163] We cloned 41 of the 43 A-type miRNAs, of which 13 miRNAs were not present on the original microarray but only computationally predicted, as well as the D-type miRNAs. For 11 of the predicted miRNA precursors, both 5p and 3p predicted mature miRNAs were present on the microarray and in all cases both gave significant signals. Thus, we attempted to clone both 5' and 3' mature miRNAs in all cloning attempts. For 27 of the 43 cloned miRNA, we were able to clone miRNA derived from both 5' and 3' stems. Since our cloning efforts were not exhaustive, it is possible that more of the miRNA precursors encode both 5' and 3' mature miRNAs.
[0164] Many of the cloned miRNAs have shown heterogeneity at the 3' end as observed in many miRNA cloning studies (Lagos-Quintana 2001, 2002, 2003) (Poy 2004). Interestingly, we also observed heterogeneity at the 5' end for a significant number of the cloned miRNAs. This heterogeneity seemed to be somewhat more prevalent in 5'-stem derived miRNAs (9) compared to 3'-stem derived miRNAs (6). In comparison, heterogeneity at the 3' end was similar for both 3' and 5'-stem derived miRNAs (19 and 13, respectively). The 5' heterogeneity involved mainly addition of one nucleotide, mostly C or A, but in one case there was an addition of 3 nucleotides. This phenomenon is not specific to the miRNAs in the chromosome 19 cluster. We have observed it for many additional cloned miRNAs, including both known miRNAs as well as novel miRNAs from other chromosomes (data not shown).
[0165] To further examine the expression of the miRNAs of Example 4, we used Northern blot analysis to profile miRNA expression in several tissues. Northern blot analysis was performed using 40 g of total RNA separated on 13% denaturing polyacrylamide gels and using 32P end labeled oligonucleotide probes. The oligonucleotide probe sequences were 5'-ACTCTAAAGAGAAGCGCTTTGT-3'(SEQ ID NO:760617) (A19-3p, NCBI: HSA-MIR-RG-21) and 5'-ACCCACCAAAGAGAAGCACTTT-3'(SEQ ID NO:760618) (A24-3p, NCBI: HSA-MIR-RG-27). The miRNAs were expressed as ˜22 nucleotide long RNA molecules with tissue specificity profile identical to that observed in the microarray analysis (FIG. 5-A).
[0166] In order to determine how the MC19-1 cluster is transcribed. A survey of the ESTs in the region identified only one place that included ESTs with poly-adenylation signal and poly-A tail. This region is located just downstream to the A43 precursor. The only other region that had ESTs with poly-adenylation signal is located just after mir-373, suggesting that mir-371,2,3 are on a separate transcript. We performed initial studies focusing on the region around mir-A43 to ensure that the region is indeed transcribed into poly-adenylated mRNA. RT-PCR experiments using primers covering a region of 3.5 kb resulted in obtaining the expected fragment (FIG. 5-B). RT-PCR analysis was performed using 5 g of placenta total RNA using oligo-dT as primer. The following primers were used to amplify the transcripts: f1:
5'-GTCCCTGTACTGGAACTTGAG-3'(SEQ ID NO:760619) ; f2:
5'-GTGTCCCTGTACTGGAACGCA-3'(SEQ ID NO:760620) ; r1:
5'-GCCTGGCCATGTCAGCTACG-3'(SEQ ID NO: 760621) ; r2:
5'-TTGATGGGAGGCTAGTGTTTC-3'(SEQ ID NO: 760622); r3:
[0167] 5'-GACGTGGAGGCGTTCTTAGTC-3'(SEQ ID NO: 760623); and r4: 5'-TGACAACCGTTGGGGATTAC-3' (SEQ ID NO: 760624). The authenticity of the fragment was validated by sequencing. This region includes mir-A42 and mir-A43, which shows that both miRNAs are present on the same primary transcript.
[0168] Further information on the transcription of the cluster came from analysis of the 77 ESTs located within it. We found that 42 of the ESTs were derived from placenta. As these ESTs are spread along the entire cluster, it suggested that the entire cluster is expressed in placenta. This observation is in-line with the expression profile observed in the microarray analysis. Thus, all miRNAs in the cluster may be co-expressed, with the only exception being the D-type miRNAs which are the only miRNAs to be expressed in HeLa cells. Interestingly, none of the 77 ESTs located in the region overlap the miRNA precursors in the cluster. This is in-line with the depletion of EST representation from transcripts processed by Drosha.
[0169] Examination of the microarray expression profile revealed that miRNAs D1/2, A12, A21, A22, and A34, have a somewhat different expression profile reflected as low to medium expression levels in several of the other tissues examined. This may be explained by alternative splicing of the transcript(s) encoding the miRNAs or by the presence of additional promoter(s) of different tissues specificity along the cluster.
[0170] Comparison of the expression of 3p and 5p mature miRNAs revealed that both are expressed for many miRNA precursors but in most cases at different levels. For most pre-miRNAs the 3 p miRNAs are expressed at higher levels then the 5p miRNAs. However, in 6 cases (mir-D1,2, mir-A1, mir-A8, mir-A12, mir-A17 and mir-A33) both 3p and 5p miRNAs were expressed at a similar level, and in one case (mir-A32) the 5 p miRNA was expressed at higher levels then the 3 p miRNA.
[0171] Comparison of the sequences from all four types of predicted miRNAs of Example 4 to that of other species (chimp, macaque, dog, chicken, mouse, rat, drosophila, zebra-fish, fungi, c. elegans) revealed that all miRNAs in the cluster, and in fact the entire region, are not conserved beyond primates. Interestingly, homologues of this region do not exist in any other genomes examined, including mouse and rat. Thus, this is the first miRNA cluster that is specific to primates and not generally shared in mammals. Homology analysis between chimp and human show that all 35 repeats carrying the A-type miRNAs are contiguous between the two species. Furthermore, the entire cluster seems to be identical between human and chimp. Thus, the multiple duplications leading to the emergence of the MC19-1 cluster must have occurred prior to the split of chimp and human and remained stable during the evolution of each species. It should be noted that human chromosome 19 is known to include many tandemly clustered gene families and large segmental duplications (Grimwood et al, 2004). Thus, in this respect the MC19-1 cluster is a natural part of chromosome 19.
[0172] In comparison, the MC14-1 cluster is generally conserved in mouse and includes only the A7 and A8 miRNAs within the cluster are not conserved beyond primates (Seitz 2004). In contrast all miRNAs in the MC19-1 cluster are unique to primates. A survey of all miRNAs found in Sanger revealed that only three miRNA, mir-198, mir-373, and mir-422a, are not conserved in the mouse or rat genomes, however, they are conserved in the dog genome and are thus not specific to primates. Interestingly, mir-371 and mir-372, which are clustered with mir-373, and are located 25 kb downstream to the MC19-1 cluster, are homologous to some extent to the A-type miRNAs (FIG. 4), but are conserved in rodents.
[0173] Comparison of the A-type miRNA sequences to the miRNAs in the Sanger database revealed the greatest homology to the human mir-302 family (FIG. 4-C). This homology is higher than the homology observed with mir-371,2,3. The mir-302 family (mir-302a, b, c, and d) are found in a tightly packed cluster of five miRNAs (including mir-367) covering 690 nucleotides located in the antisense orientation in the first intron within the protein coding exons of the HDCMA18P gene (accession NM-- 016648). No additional homology, apart from the miRNA homology, exists between the mir-302 cluster and the MC19-1 cluster. The fact that both the mir-371, 2, 3 and mir-302a, b, c, d are specific to embryonic stem cells is noteworthy.
[0174] Differential Expression of miRNAs
[0175] Using chip expression methods similar to those described in 0, microarray images were analyzed using Feature Extraction Software (Version 7.1.1, Agilent). Table 2 shows the ratio of disease related expression ("R") compared to normal tissues. Table 2 also shows the statistical analysis of the normalized signal ("RPval"). The signal of each probe was set as its median intensity. Signal intensities range from background level of 400 to saturating level of 66000. 2 channels hybridization was performed and Cy3 signals were compared to Cy5 signals, where fluor reversed chip was preformed (normal vs. disease), probe signal was set to be its average signal. Signals were normalized by dividing them with the known miRNAs average signals such that the sum of known miRNAs signal is the same in each experiment or channel. Signal ratios between disease and normal tissues were calculated. Signal ratio greater than 1.5 indicates a significant upregulation with a P value of 0.007 and signal ratio grater than 2 has P value of 0.003. P values were estimated based on the occurrences of such or greater signal ratios over duplicated experiments.
[0176] The differential expression analysis in Table 2 indicates that the expression of a number of the miRNAs are significantly altered in disease tissue. In particular, the MC19-1 miRNAs of Example 4 are differentially expressed in prostate and lung cancer. The relevance of the MC19-1 miRNAs to cancer is supported by the identification of a loss of heterozygosity within the MC19-1 region in prostate cancer derived cells (Dumur et al. 2003).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application contains a lengthy "Sequence Listing" section. A copy of the "Sequence Listing" is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120094374A1). An electronic copy of the "Sequence Listing" will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).