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WO2008053909A1 - Agent for controlling growth and/or differentiation of mesenchymal stem cell - Google Patents

Agent for controlling growth and/or differentiation of mesenchymal stem cell Download PDF

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Publication number
WO2008053909A1
WO2008053909A1 PCT/JP2007/071177 JP2007071177W WO2008053909A1 WO 2008053909 A1 WO2008053909 A1 WO 2008053909A1 JP 2007071177 W JP2007071177 W JP 2007071177W WO 2008053909 A1 WO2008053909 A1 WO 2008053909A1
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WIPO (PCT)
Prior art keywords
nucleic acid
seq
differentiation
mesenchymal stem
base sequence
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Ceased
Application number
PCT/JP2007/071177
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French (fr)
Japanese (ja)
Inventor
Yoji Yamada
Tatsuya Miyazawa
Tetsuo Yoshida
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KH Neochem Co Ltd
Kyowa Kirin Co Ltd
Original Assignee
Kyowa Hakko Kogyo Co Ltd
Kyowa Hakko Kirin Co Ltd
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Publication of WO2008053909A1 publication Critical patent/WO2008053909A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/65—MicroRNA

Definitions

  • the present invention relates to an inhibitor of proliferation and / or differentiation of mesenchymal stem cells, an inhibitor of expression of a target gene of a mouthpiece RNA that is one of nucleic acids, and a method of inhibiting proliferation and / or differentiation of mesenchymal stem cells.
  • MicroRNA target gene expression suppression method mesenchymal stem cell proliferation and / or differentiation inhibitor screening method, mesenchymal stem cell proliferation and / or differentiation promoter, mesenchymal stem cell proliferation and / or The present invention also relates to a method for promoting differentiation, a diagnostic agent for a disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells, and a therapeutic agent for a disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells.
  • MicroRNA a kind of nucleic acid, is a small non-coding single-stranded RNA of about 22 nucleotides that is not translated into protein, and has been confirmed to exist in many organisms including humans (non-native). Patent Documents 1 and 2).
  • MicroRNAs are generated from genes that are transcribed into single or clustered microRNA precursors. That is, first, primary-microRNA (pri-miRNA), a primary transcript, is transcribed from the gene, and then for stepwise processing from pri-miRNA to mature microRNA! /, From pri-miRNA Approximately 70 base pr ecursor-microRNA (pre-miRNA) having a characteristic hairpin structure is produced. Furthermore, mature microRNA is produced from pre-miRNA by Dicer-mediated processing (Non-patent Document 3).
  • pri-miRNA primary-microRNA
  • pre-miRNA a primary transcript
  • Mature microRNAs are thought to be involved in post-transcriptional regulation of gene expression by binding complementarily to the target mRNA and suppressing translation of the mRNA, or by degrading the mRNA.
  • miRBase http: microrna.sanger.ac.uk/
  • miR-181 those that are known for their physiological functions include miR-181 (Non-patent Document 4) involved in blood cell differentiation and miR- involved in secretion of insulin.
  • Non-Patent Document 5 miR_143 that suppresses the growth of cancer cells, miR-145 (Non-patent document 11), miR-372 that promotes cancer cell growth, miR_373 (Non-patent document 12), etc. are only some of them, and many of them have unclear physiological activity.
  • studies using nematodes and Drosophila have revealed that microRNAs play various important roles in the development and differentiation of organisms, especially in relation to human diseases. There have been reports suggesting a deep relationship with cancer (Non-patent Document 6).
  • microRNA For identification of microRNA, there are a method of cloning small RNA from a cell, a method of using bioinformatics from genome sequence information, and the like. In order to be registered as a microRNA in miRBase, both information related to expression and information related to biosynthesis and structure are required, and it is not recognized as a microRNA simply by predicting the structure from genomic sequence information (Non-patent Documents). 7).
  • Mesenchymal stem cells are known as pluripotent stem cells that are present in mammalian bone marrow, adipose tissue, umbilical cord blood, and the like and differentiate into adipocytes, chondrocytes, bone cells, and the like. Because of their pluripotency, mesenchymal stem cells are attracting attention as a transplant material for regenerative medicine of many tissues such as bone, cartilage, tendon, muscle, fat, and periodontal tissue! Non-patent document 8).
  • Mesenchymal stem cells can be differentiated into specific cells in vitro by the addition of drugs, cytodynamic force, etc.
  • drugs cytodynamic force, etc.
  • 1-methyl-3-isobutylxanthine, dexamethasone, insulin and indomethacin can be differentiated into adipocytes.
  • Non-patent Document 9 Non-patent Document 9
  • Non-patent Document 10 “Science J, 2001, 294, p.853-858
  • Non-Patent Document 2 “Cell”, 2003, 113, 673-676
  • Non-Patent Document 3 "Nature Reviews Genetics", 2 004, volume 5, .522-531
  • Non-Patent Document 4 "Science”, 2004, 303, .83-86
  • Non-Patent Document 5 “Nature”, 2004, 432, 226-230
  • Non-Patent Document 6 “Nature Reviews Cancer”, 2006, Vol. 6, 259-269
  • Non-Patent Document 7 “NA”, 2003, Vol. 9, 277-279
  • Non-Patent Document 8 “Gene Medicine”, 2000, vol. 4, p. 58-61
  • Non-Patent Document 9 “Science”, 1999, 284, ⁇ ⁇ 143-147
  • Non-Patent Document 10 "Experimental Medicine", 2002, 20, p. 2459-2464
  • Non-Patent Document 11 “Oncology Report”, 2006, 16, 845-85
  • Non-Patent Document 12 “Cell”, 2006, 124, p. 1169-1181
  • microRNAs that are expressed in various human organs ! By identifying microRNAs that are expressed in various human organs !, analyzing their functions, and elucidating their relationship with diseases, new therapeutics and diagnostics may be developed. Expected In particular, the discovery of microRNAs acting on mesenchymal stem cells leads to elucidation of the functions of differentiation and proliferation in mesenchymal stem cells, and isolation, culture, and mesenchymal stem cells from mesenchymal stem cells It can be expected that this will lead to the development of differentiation control methods for specific cells and new therapies using differentiation control.
  • An object of the present invention is to obtain a microphone RNA group expressed in mesenchymal stem cells and provide a method for using them.
  • the present invention relates to the following (1) to (40).
  • nucleic acid comprising a base sequence having 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 93
  • nucleic acid comprising the second to eighth base sequences of the base sequence represented by any of SEQ ID NOs:! to 5
  • a double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid
  • Double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid that hybridizes with the nucleic acid under stringent conditions
  • nucleic acid comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence represented by any of SEQ ID NOS: 94 to 201
  • nucleic acid that hybridizes with a complementary strand of a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201 under stringent conditions.
  • a mesenchymal stem cell proliferation and / or differentiation inhibitor comprising the vector expressing the nucleic acid according to (1) as an active ingredient.
  • siRNA is a siRNA whose target sequence is a nucleotide sequence represented by SEQ ID NOs: 202 to 205!
  • An ADD3 gene expression inhibitor comprising as an active ingredient a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 97.
  • ADD3 gene expression inhibitor comprising as an active ingredient a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid .
  • An ADD3 gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.
  • a CBFB gene expression inhibitor comprising as an active ingredient a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 97.
  • (13) CBFB gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid .
  • a CBFB gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising a nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.
  • the substance that suppresses the expression of the ADD3 gene or the CBFB gene is a nucleic acid (the method for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to 18). (20) The method for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to (19), wherein the nucleic acid is siRNA.
  • siRNA is a siRNA whose target sequence is the base sequence represented by any of SEQ ID NOs: 202 to 205.
  • (22) A method for suppressing expression of a target gene of the nucleic acid, using the nucleic acid according to (1).
  • a method for suppressing the expression of the ADD3 gene which uses a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the base sequence represented by SEQ ID NO: 97.
  • a method for suppressing ADD3 gene expression comprising using a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid.
  • a method for suppressing ADD3 gene expression comprising using a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.
  • a method for suppressing the expression of a CBFB gene which uses a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the base sequence represented by SEQ ID NO: 97.
  • a method for inhibiting CBFB gene expression comprising using a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.
  • (29) A method for screening for an agent for suppressing the proliferation and / or differentiation of mesenchymal stem cells using the expression of the nucleic acid according to (1) as an index.
  • (31) A method for screening for an agent for promoting the proliferation and / or differentiation of mesenchymal stem cells using the expression of the nucleic acid according to (1) as an index.
  • (32) A method for screening an agent for promoting proliferation and / or differentiation of mesenchymal stem cells using the expression of the expression of the target gene of the nucleic acid according to (1) as an index.
  • (35) A method for promoting the proliferation and / or differentiation of mesenchymal stem cells using the substance that suppresses the expression of a nucleic acid according to (1).
  • the mesenchymal stem cell proliferation and / or differentiation inhibitor according to any one of (1) to (7) or the mesenchymal stem cell proliferation and / or differentiation promotion according to (33) or (34) A therapeutic agent for diseases caused by abnormal proliferation and / or differentiation of mesenchymal stem cells, which contains the agent as an active ingredient.
  • a mesenchymal stem cell proliferation and / or differentiation inhibitor a microRNA target gene expression inhibitor, a mesenchymal stem cell proliferation and / or differentiation suppression method, and a microphone mouth RNA target gene Expression suppression method, mesenchymal stem cell proliferation and / or differentiation inhibitor screening method, mesenchymal stem cell proliferation and / or differentiation promoting agent, mesenchymal stem cell proliferation and / or differentiation promoting method, mesenchyme It is possible to provide a diagnostic agent for diseases caused by abnormal stem cell proliferation and / or differentiation and a therapeutic agent for diseases caused by abnormal mesenchymal stem cell proliferation and / or differentiation.
  • Fig. 1 shows changes in microRNA expression during hMSC osteoblast differentiation.
  • A shows hsa_miR-100
  • B shows hsa_miR-145
  • C shows hsa_miR-210
  • D shows hsa-miR-4 22b.
  • the horizontal axis represents the number of culture days
  • the vertical axis represents the relative expression level, where 1.0 is the expression level of each microRNA expressed in hMSC before differentiation induction.
  • nucleic acid used for the agent for suppressing proliferation and / or differentiation of mesenchymal stem cells of the present invention examples include the following nucleic acids.
  • the nucleic acid is preferably microRNA or a derivative thereof, a microRNA precursor or a derivative thereof and a double-stranded nucleic acid (hereinafter also referred to as a nucleic acid used in the present invention).
  • nucleic acid comprising a base sequence having 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 93
  • nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid comprising the nucleotide sequence represented by any of SEQ ID NOs: 1 to 93
  • nucleic acid comprising the second to eighth base sequences of the base sequence represented by any of SEQ ID NOs:! to 5
  • a double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid
  • Double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid that hybridizes with the nucleic acid under stringent conditions
  • nucleic acid comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence represented by any of SEQ ID NOS: 94 to 201
  • nucleic acid that hybridizes with a complementary strand of a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201 under stringent conditions.
  • the microRNA is a single-stranded RNA present in a cell, and the The surrounding genomic sequence including the sequence has RNA that can form a hairpin structure, and it means RNA that can cut out any one strand of the hairpin.
  • the length of the microRNA is preferably 15 to 28 bases, more preferably 16 to 28 bases, more preferably 16 to 26 bases, and particularly preferably 16 to 24 bases.
  • MicroRNA binds complementarily to its target mRNA, suppresses mRNA degradation or translation, and controls post-transcriptional gene expression. Examples of the microRNA used in the present invention include human microRNA having a base sequence represented by any of SEQ ID NOs:! -5.
  • the base represented by SEQ ID NOs: 6 to 65 which is an ortholog of the human microRNA It is possible to raise nucleic acids consisting of sequences.
  • the human microRNA ortholog of SEQ ID NO: 1 is SEQ ID NO: 6-20
  • the human microRNA ortholog of SEQ ID NO: 2 is SEQ ID NO: 21-40
  • the human microRNA ortholog of SEQ ID NO: 3 is a sequence No. 4;!
  • the human microRNA ortholog of SEQ ID NO: 4 is a nucleic acid comprising the nucleotide sequences represented by SEQ ID NOs: 53-63
  • the human microRNA of SEQ ID NO: 5 is represented by SEQ ID NOs: 64-65, respectively.
  • the ability to raise S in addition, as a mechanism by which microRNA suppresses translation of mRNA of its target gene, mRNA having a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is used as a microRNA target gene. It is known that its translation is suppressed by microRNA [Current Biology, 15, R458-R460 (2005)].
  • the microRNA having the same base sequence has the same function by suppressing translation of the same mRNA.
  • Specific examples of the microRNA include SEQ ID NO: 66 to 67 for the microRNA of SEQ ID NO: 1, and SEQ ID NO: 68 to 92 for the microRNA of SEQ ID NO: 2, and the microRNA of SEQ ID NO: 5.
  • RNA it refers to the ability to raise a nucleic acid consisting of the base sequence represented by SEQ ID NO: 93.
  • the microRNA precursor is a nucleic acid having a length of about 50 to about 200 bases, more preferably about 70 to about 100 bases including the microRNA used in the present invention, and forms a hairpin structure.
  • MicroRNA is produced from the microRNA precursor through processing by a protein called Dicer.
  • Micro RN used in the present invention As the A precursor, for example, SEQ ID NO: 94 to 95 for the human microRNA of SEQ ID NO: 1, SEQ ID NO: 96 for the human microRNA of SEQ ID NO: 2, against the human microRNA of SEQ ID NO: 3 Is the nucleotide sequence IJ force represented by SEQ ID NO: 98 for the human microRNA of SEQ ID NO: 97 and SEQ ID NO: 4, and SEQ ID NO: 99 for the human microRNA of SEQ ID NO: 5, respectively. That power S. Furthermore, the ortholog microRNA precursor functions in the same manner as the human microRNA precursor, and can therefore be used as a microRNA precursor for use in the present invention.
  • microRNA precursor to an ortholog of the human microRNA of SEQ ID NO: 1 represented by any one of SEQ ID NOS: 6-20 IJ numbers 100-; 126, SEQ ID NO: 2; A human microRNA ortholog of SEQ ID NO: 3 represented by any one of SEQ ID NO: 127 to 147, SEQ ID NO: 4;! -52 as a microRNA precursor to a human microphone RNA ortholog of SEQ ID NO: 2 represented by As a microRNA precursor for human microRNA ortholog of SEQ ID NO: 4 represented by any one of SEQ ID NO: 148-159 and SEQ ID NO: 53-63 as a microRNA precursor for IJ No.
  • a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 173 to 174, SEQ ID NOs: 68 to 92
  • a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 175 to 200
  • SEQ ID NO: 93 represented by SEQ ID NO: 201
  • a nucleic acid consisting of a base sequence can be mentioned.
  • a nucleic acid having 90% or more identity with the base sequence represented by any of SEQ ID NOs:! To 201 is BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA.
  • FASTA FASTA
  • at least 90% or more of the nucleic acid consisting of the nucleotide sequence represented by any of SEQ ID NOs: 1-201 is preferred.
  • stringent conditions are 7.5 mL, 1 M Na HPO (pH 7.2) 0.6 mL, 10% SDS 21 mL, 50x Denhardt's solution 0.6 mL, 10 mg on the membrane blotted on one strand.
  • the nucleic acid may be any molecule in which nucleotides and molecules having functions equivalent to the nucleotides are polymerized.
  • RNA which is a polymer of ribonucleotides, and deoxyribonucleotides.
  • the polymer include DNA, RNA and a mixture of RNA and nucleotide, and nucleotide polymers including nucleotide analogs, and may be nucleotide polymers including nucleic acid derivatives. It may be a nucleic acid or a double-stranded nucleic acid.
  • MicroRNA or a derivative thereof, a microRNA precursor or a derivative thereof are also included in the nucleic acid of the present invention.
  • a nucleotide analog is used to increase the affinity with a complementary strand nucleic acid in order to improve or stabilize nuclease resistance compared to RNA or DNA.
  • the sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting an arbitrary chemical structural substance to a part or all of the chemical structure of the sugar of the nucleotide.
  • Substituted nucleotide analogs nucleotide analogs substituted with 2'-0- [2- (guanidinium) ethyl] ribose, nucleotide analogs substituted with 2 '0-fluororibose, cross-linked to the sugar moiety
  • Bridged Nucleic Acid (BNA) having two circular structures by introducing a structure, more specifically, a 2′-position oxygen atom and a 4′-position carbon
  • LNA Locked Nucleic Acid
  • Ethylene bridged nucleic acid PNA
  • PNA peptides Nucleic acid
  • OPNA Oxypeptide nucleic acid
  • PRNA peptide ribonucleic acid
  • Phosphodiester bond-modified nucleotide analogs are those in which any chemical substance is added to part or all of the chemical structure of a phosphodiester bond of a nucleotide!
  • nucleotide analogues substituted with phosphorothioate bonds nucleotide analogues substituted with ⁇ 3'_ ⁇ 5 'phosphoramidate linkages, and the like can be mentioned.
  • modified sugars include oligonucleotide derivatives substituted with 2'-0_propyl ribose, oligonucleotide derivatives substituted with 2'-methoxyethoxyribose, 2, -0-methyl Oligonucleotide derivatives substituted with ribose, oligonucleotide derivatives substituted with 2, -0-methoxyethyl ribose, oligonucleotide derivatives substituted with 2, -0- [2- (guanidinium) ethyl] ribose, 2 'It is possible to raise oligonucleotide derivatives substituted with -0-fluororibose, and the modified phosphate group is an oligonucleotide in which the phosphate diester bond in the oligonucleotide is converted to a phosphoroate bond.
  • Nucleotide derivatives and phosphodiester bonds in oligonucleotides are ⁇ 3'_ ⁇ 5 'phosphoramidate bonds And the like. [Cell engineering, 16, 1463-1473 (1997)] [RNAi method and antisense method, Kodansha (2005)].
  • the nucleic acid derivative is used for improving nuclease resistance, stabilizing, increasing affinity with a complementary strand nucleic acid, increasing cell permeability, or visualization compared to nucleic acid.
  • 5 'polyamine addition derivative cholesterol addition derivative, steroid addition derivative, bile acid addition derivative, vitamin addition derivative, Cy5 Addition derivatives, Cy3 addition derivatives, 6-FAM addition derivatives, biotin addition derivatives, and the like.
  • the microRNA derivative may be any molecule in which a molecule having a function equivalent to that of the microRNA is a polymerized molecule.
  • the DNA polymer include DNA, RNA and DNA mixed polymers, and nucleotide polymers including nucleotide analogs.
  • nucleotide polymers including nucleic acid derivatives may be used. Can be single-stranded or double-stranded nucleic acid! /.
  • the derivative of the microRNA precursor may be any molecule in which a molecule having a function equivalent to that of the microRNA precursor is polymerized.
  • RNA or deoxy that is a polymer of ribonucleotides
  • the ribonucleotide polymer include DNA, a polymer in which RNA and DNA are mixed, and a nucleotide polymer including a nucleotide analog, and may be a nucleotide polymer including a nucleic acid derivative. It can be a single-stranded nucleic acid or a double-stranded nucleic acid.
  • Mesenchymal stem cells are present in mesenchymal tissues such as bone marrow, adipose tissue, umbilical cord blood, endometrium, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, pulp, and tooth germ.
  • mesenchymal tissues such as bone marrow, adipose tissue, umbilical cord blood, endometrium, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, pulp, and tooth germ.
  • mesenchymal tissues such as bone marrow, adipose tissue, umbilical cord blood, endometrium, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, pulp, and tooth germ.
  • mesenchymal tissues such as bone marrow, adipose tissue, umbilical cord blood, endometrium, dermis, skeletal muscle, periosteum, dental folli
  • the method for producing the microRNA or derivative thereof, microRNA precursor or derivative thereof, or double-stranded nucleic acid used in the present invention is not particularly limited, and is a method using a known chemical synthesis or an enzymatic transcription method. Etc. can be manufactured. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, and phosphorotriester method. For example, the synthesis is performed using an ABI3900 high-throughput nucleic acid synthesizer (Applied by Systems). That power S.
  • a transcription method using a typical phage RNA polymerase for example, T7, ⁇ 3, or SP6 RNA polymerase, using a plasmid or DNA having the target nucleotide sequence as a cage type S I'll do it.
  • microRNA or microRNA precursor used in the present invention
  • the presence of microRNA or microRNA precursor in a sample is detected.
  • a method for detecting the expression of microRNA or microRNA precursor used in the present invention the presence of microRNA or microRNA precursor in a sample is detected.
  • a sample is detected.
  • Northern hybridization for example, (1) Northern hybridization, (2) Dot blot hybridization, (3) In situ hybridization, (4) Quantification PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay, and the like.
  • microRNA in a sample or or Any method can be used as long as it can detect a mutation in the base sequence of the microRNA precursor.
  • a heterogeneous complex formed by hybridization of a nucleic acid having a non-mutated base sequence and a nucleic acid having a mutant base sequence examples thereof include a method for detecting a double strand, and a method for detecting the presence or absence of mutation by directly sequencing a base sequence derived from a specimen.
  • microRNA used in the present invention or a derivative thereof, a microRNA precursor or a derivative thereof, or a vector expressing a double-stranded nucleic acid is introduced into a cell and transcribed to biosynthesize the nucleic acid.
  • PCDNA6.2-GW / miR Invitrogen
  • pSilencer 4.1-CMV Ambion
  • pSINsi-hHl DNA Takara Bio
  • pSINsi_hU6 DNA Takara Bio
  • pEN TR / U6 manufactured by Invitrogen
  • the target gene is a nucleic acid base sequence consisting of several bases recognized by the microRNA used in the present invention, and a base sequence in which translation of mRNA having the base sequence is suppressed by the microRNA.
  • a gene with! The base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is that translation of mRNA having the base sequence is suppressed by the microRNA [Current Biology, 15, R458-R460 (2005)], a base sequence complementary to the 2-8th base sequence on the 5 ′ end side of the microRNA used in the present invention can be listed as a base sequence to be suppressed by the microRNA. .
  • a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA (hereinafter also referred to as the target base sequence of microRNA) is prepared, and the 3'UTR of human mRNA is prepared.
  • salt It is possible to determine by selecting a mRNA containing a completely identical sequence from a base sequence group by a method such as character string search.
  • the 3'UTR base sequence group of human mRNA is created using the genome sequence and gene position information that can be obtained from UCSC Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway) Specific examples of microRNA target genes represented by SEQ ID NOs:!
  • To 5 include US National Biotechnology ⁇ ⁇ 'Gare Information Center National Center for Biotechnology Information (NCBI) En treGene database (http://www.ncbi.nlm.nih.gov/Elntrez/) ⁇ (Recognized! /, the name ij (Official Symbol and Gene ID) The name of the gene used was the name of the EntreGene database as of March 2006. Also, the human microRNA ortholog and the human microRNA included in the present invention as the microRNA are used.
  • the target gene of RNA is the same as the target gene of the human microRNA, specifically, the target gene of a nucleic acid comprising the base sequence represented by any one of SEQ ID NOs: 6-20 and 66-67 Is the same gene as the target gene of the microRNA of SEQ ID NO: 1 in Table 1. Further, as a target gene of a nucleic acid consisting of the base sequence represented by any of SEQ ID NOS: 21-40 or 68-92 Can be the same gene as the target gene of the microRNA of SEQ ID NO: 2 in Table 1.
  • the target gene of the nucleic acid consisting of the base sequence represented by any of SEQ ID NO: 4 In Table 1, the same gene as the target gene of the mitochondrial RNA of SEQ ID NO: 3 can be mentioned, and the target gene of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOS: 53 to 63 is as follows: In Table 1 The same gene as the target gene of the microRNA of SEQ ID NO: 4. In addition, the target gene of a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 64 to 65, 93 is In 1 the same gene as the target gene of the microRNA of SEQ ID NO: 5 can be mentioned.
  • Fine d (mU) U0I-(t56Z9g) SJ, VD- (6it6) IWtJV- '(lSSi) S; WZ: (mU)
  • V8 (i98sz) s eoatg dZii) ia; (999is sv: (66)
  • VI3 ⁇ 4 (mil) INS3S: (I8CIl) 893as: (OliS SSdd: OZ) IWIV: (6 IlI) S3A
  • V901KHW1 (ZZ9) m-(6898) SDOV: (Sf69) XlW: ( ⁇ ⁇ 9) C1VIS: (Ui9) 991V
  • nsM ( ⁇ s) id:) iv thigh: (6onot)
  • IDDHV (6Z9) Image I: (t ⁇ ZS2) 09S0VVIS: (S06Z) VZ: 6H0OHd: (
  • UVZilOd (Z ⁇ W m- (8Z86) ld33MV: (S 86) ZaS W:
  • zi m- (mm) is ⁇ nd '(98t "ref: (oson) sw fine: (n 3 ⁇ : (
  • VVIX mU- (00iZS) 009IVVI3 ⁇ 4: (8tl6) ri3N: (Z) ⁇ SV: (08
  • the ADD3 gene refers to a human adducin3 (g ⁇ ma) gene contained in the base sequence represented by SEQ ID NO: 206, and an ortholog that is a homologous gene of another species corresponding thereto.
  • the CBFB gene refers to a human core-binding factor, beta subnit (CBFb) gene contained in the base sequence represented by SEQ ID NO: 207, and orthologs that are homologous genes of other species.
  • the substance that suppresses the expression of the target gene of the microRNA of the present invention may be any substance as long as it has an activity of suppressing the expression of the mRNA of the target gene of the microRNA.
  • SiRNA is particularly preferred.
  • activity to suppress expression Sex refers to (l) the activity of suppressing the translation of mRNA, and (2) the activity of cleaving or degrading the mRNA, resulting in a decrease in the amount of protein translated from the mRNA.
  • siRNA for the ADD3 gene can be exemplified, and more preferably, the base sequence represented by any one of SEQ ID NOs: 202 to 203 is used as the target sequence.
  • siRNA against the CBFB gene can be raised, and more preferably, the siRNA having the base sequence represented by any of SEQ ID NOs: 204 to 205 as a target sequence Can give.
  • the disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells is not particularly limited as long as it is a disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells.
  • specific examples include cancer, osteogenesis dysplasia, chondrogenesis dysplasia, and glucoseuria.
  • the method for obtaining mesenchymal stem cells from the bone marrow is not particularly limited as long as it is a safe and efficient method.
  • the method described in SE Haynesworth et al. Bone, 13, 81 (199 2) Can be given.
  • bone marrow cells containing 10% urine fetal serum (FBS) a -MEM (a -modified MEM), DMEM (Dulbecco's modified MEM), I MDM (Isocove's modified Dulbecco's
  • FBS urine fetal serum
  • a -MEM a -modified MEM
  • DMEM Dulbecco's modified MEM
  • I MDM Isocove's modified Dulbecco's
  • a bone marrow cell solution is obtained by suspending in a cell culture medium such as medium).
  • a method for isolating mesenchymal stem cells from bone marrow cell fluid other cells mixed in the bone marrow cell fluid such as blood cells, hematopoietic stem cells, hemangioblasts
  • the fountain fibroblasts can be removed without particular limitation, and examples thereof include the method described in MF Pittenger et al.
  • the bone marrow cell solution can be layered on Percoll with a density of 1.073 g / ml, then centrifuged at 1,100 X g for 30 minutes to isolate the cells at the interface as mesenchymal stem cells. Also, add 10X PBS to the bone marrow cell solution and mix with Percoll diluted to 9/10, add the same volume and mix, then centrifuge for 30 minutes at 20,000 X g, and collect the fraction of density 1.075-1060 Cells can also be isolated as mesenchymal stem cells.
  • mesenchymal stem cells derived from human bone marrow can be purchased from Cambrex and Takara Bio.
  • the method for obtaining mesenchymal stem cells from the umbilical cord is not particularly limited as long as it is an efficient method, and examples thereof include the method described in Stem Cells, 21, 105-110 (2003).
  • a force neulet is inserted into both ends of the umbilical vein and washed with an appropriate buffer, for example, EBSS (Earle's balanced salt solution).
  • Antibiotics are added to a 199 medium containing a proteolytic enzyme, for example, 0.1% collagenase, injected into a blood vessel, and incubated at 4 to 40 ° C, preferably 37 ° C for 1 to 60 minutes.
  • DMEM medium DMEM-LG, Gibco
  • glucose 20 mM HEPES
  • 100 units / ml penicillin 100 units / ml penicillin.
  • Suspend in medium supplemented with 100 ⁇ g / ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 10% FBS.
  • the culture flask is inoculated at a cell density of 10 2 to 10 6 m 2 and cultured under conditions of 37 ° C. and 5% CO 2.
  • Mesenchymal stem cells can be obtained by changing the medium every 1 to 7 days and continuing the culture for! ⁇ 3 weeks.
  • the method for obtaining mesenchymal stem cells from the endometrium is not particularly limited as long as it is a safe and efficient method.
  • Am. J. Pathol. 163, 2259-2269 ( 200 3).
  • Human endometrial tissue excised by surgery is cut into small pieces and cultured in a culture medium, preferably ⁇ _ ⁇ , DMEM, IMDM, etc. 1-20% animal-derived serum, preferably 5-10% Incubate in medium supplemented with FBS.
  • Antibiotics such as penicillin and streptomycin may be added to the medium.
  • collagen-degrading enzymes such as type 3 collagenase and deoxyribonuclease
  • DNA-degrading enzyme such as zase I
  • Individual endometrial glands can be isolated while observing under a microscope and cultured in an appropriate culture vessel such as 24-well culture dish at 37 ° C and 5% C 0 to obtain mesenchymal stem cells. Can be obtained.
  • the method for obtaining mesenchymal stem cells from teeth, tooth embryos, and tissues around teeth is not particularly limited as long as it is a method that can be obtained safely and efficiently.
  • Human teeth may be milk teeth or permanent teeth such as incisors, canines, premolars, and molars.
  • the periodontal ligament is carefully separated from the surface of the extracted third molar (wisdom tooth) root, and proteolytic enzymes such as collagenase, trypsin, pronase, elastase, dispase, and hyaluronidase are used. Perform the quenching reaction at 37 ° C for 1 hour.
  • Mesenchymal stem cells can be obtained by removing tissue residues using a strainer, mesh, filter, etc.
  • the surface of the extracted third molar (wisdom tooth) is washed with PBS, and then the cementum and enamel joints are cut to expose the medulla, and the pulp tissue is carefully removed from the crown and root.
  • Mesenchymal stem cells can be obtained by separating and treating with proteolytic enzyme in the same manner as described above, and then removing the tissue residue.
  • a reporter vector having a surface antigen expressed in mesenchymal stem cells, a promoter of a gene specific to mesenchymal stem cells and an enzyme is used. And a method for isolating mesenchymal stem cells. Specifically, AC
  • H 0eC Saidopopi Yureshiyon (side population) (SP) method of concentrating the stem cells in using FACS fractionation method was an indicator extracellular Emptying of hst33342 [Journal of Experim ental Medicine, 183, 1797 -806 (1996)] can also be used to isolate stem cells.
  • cells that are SH2 positive, SH4 positive, CD29 positive, CD44 positive, CD71 positive, CD90 positive, CD 106 positive, CD 120a positive, CD124 positive, CD14 negative, CD34 negative, and CD45 negative are mesenchymal.
  • a method of separating using a cell sorter or magnetic beads [Science, 284, 14 3-147 (1999)] can also be used.
  • Examples of the medium used for culturing mesenchymal stem cells include cell culture media described in, for example, Tissue Culture Technology Fundamentals 3rd Edition, Asakura Shoten (1996), etc.
  • a cell culture medium such as ⁇ _ ⁇ , DMEM, IMDM and the like to which 1 to 20% of serum is added is preferable.
  • the culture conditions may be any conditions as long as the cells can be cultured, but the culture temperature is preferably 33 to 37 ° C.
  • the culture is preferably performed in an incubator filled with 5 to 10% CO gas.
  • the mesenchymal stem cells are preferably allowed to grow by adhering to a normal tissue culture plastic culture dish. When the cells grow on the entire culture dish, remove the medium and add the trypsin EDTA solution to suspend the cells. Suspended cells are washed with PBS or cell culture medium, diluted to 2 to 20 times with cell culture medium and freshly seeded in a culture dish, and further subcultured. be able to.
  • RNA As a method for obtaining low molecular weight RNA, specifically, 15% polyacrylamide gel electrophoresis was performed according to the method described in Jeans & Development 15, 188-200 (2000). Separation and excision of small RNA by 5 'end dephosphorylation, 3' adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, concatamerization, ligation to vector The method of cloning low molecular weight RNA and determining the base sequence of the clone, etc. can be mentioned.
  • RNA separation and excision of small RNA by 15% polyacrylamide gel electrophoresis separation and excision of small RNA by 15% polyacrylamide gel electrophoresis, 5 ′ adenylation 3′-adapter ligation Examples include a method of clotting a low molecular weight RNA and determining the base sequence of the clone through a sequence of gating, 5 ′ adapter ligation, reverse transcription, PCR amplification, concatenation, and ligation to a vector.
  • RNA can be obtained by cloning low-molecular-weight RNA and determining the base sequence by reading the base sequence of the microbeads.
  • small RNA can be obtained using a small RNA Cloning Kit (manufactured by Takara Bio Inc.).
  • RNA sequence is a microRNA can be determined by following the criteria described in RNA (RNA), 9, 277-279 (2003).
  • RNA RNA
  • 9, 277-279 (2003) RNA
  • RNA 9, 277-279 (2003).
  • a low molecular weight RNA obtained by the above method and the base sequence determined it can be carried out as follows.
  • RNAfold Nucleic Acids Research 31, 3429-3431 (2003)
  • Mfold Nucleic Acids Research 31, 3406-341 5 (2003).
  • miRBase 11570 97436265.13
  • microRNAs that are identified by the above method and expressed in mesenchymal stem cells include nucleic acids having the base sequence represented by any one of SEQ ID NOs: 1 to 5. Furthermore, as a microphone RNA having the same function as the human microRNA represented by any of SEQ ID NOs: 1 to 5, the nucleotide sequence represented by SEQ ID NOs: 6-65, which is an ortholog of the human microRNA The nucleic acid which consists of can be mention
  • mRNA having a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is It is known that it is recognized as a target gene of E.
  • Micromouth RNA having the same base sequence as the 2nd to 8th positions on the 5 ′ end of human microRNA represented by SEQ ID NOs: 1 to 5 is considered to have the same function by suppressing translation of the same mRNA.
  • a nucleic acid having a base sequence represented by SEQ ID NOs: 66 to 93 can be mentioned.
  • microRNA precursors represented by any one of SEQ ID NOs:! To 5 and expressed in mesenchymal stem cells include nucleic acids having a base sequence represented by any one of SEQ ID NOs: 94 to 99 be able to.
  • microRNA precursors represented by SEQ ID NOs: 6 to 93 include nucleic acids having the base sequences represented by SEQ ID NOs: 100 to 201.
  • RNA that is a ribonucleotide polymer based on the base sequence is identified.
  • the ability to synthesize DNA a polymer of deoxyribonucleotides. For example, based on the RNA base sequence identified in 1 above, the DNA base sequence can be determined.
  • the DNA base sequence IJ corresponding to the RNA base sequence can be uniquely determined by replacing U (uracil) contained in the RNA sequence with T (thymine).
  • a polymer in which ribonucleotides and deoxyribonucleotides are mixed or a polymer containing nucleotide analogues can be synthesized in the same manner.
  • the method for synthesizing the nucleic acid used in the present invention is not particularly limited, and the nucleic acid can be produced by a method using a known chemical synthesis or an enzymatic transcription method.
  • Known methods using chemical synthesis include the phosphoramidite method, the phosphorothioate method, and the phosphorotriate method.
  • the ability to synthesize with an ABI3900 high-throughput nucleic acid synthesizer can be mentioned.
  • the enzymatic transcription method include transcription using a typical phage RNA polymerase, for example, T7, ⁇ 3, or SP6 RNA polymerase, with a plasmid or DNA having the desired base sequence as a cage.
  • MicroRNA is a precursor of microRNA that has a hairpin structure. MicroRNA is produced through processing by a protein called Dicer, which is a kind of RNase III endonuclease, in the cytoplasm, and suppresses translation of mRNA having the target base sequence. Therefore, it is possible to detect whether or not the nucleic acid obtained based on whether or not it has the function is a microRNA.
  • Dicer a protein that is a kind of RNase III endonuclease
  • RNA force is processed by SRNaselll endonuclease.
  • a single-stranded RNA whose function is to be detected is reacted with RNaselll endonuclease and the reaction product is electrophoresed, if it has a function as a microRNA precursor, it is processed 20- By detecting a band with a length of about 25 bases, it can be detected whether it has a microRNA function.
  • RNaselll endonucl ease is not particularly limited as long as it has an activity to process a microRNA precursor, but it is preferable to use Dicer protein.
  • si-RNAse IIITM (Takara Bio), Cold Shock-DICER (Takara Bio), Recombinant Dicer Enzyme (Stratagene), BLOCK-iT Dicer RNAi Transfection Kit (Invitrogen), X- It can be obtained from treme GENE siRNA Dicer Kit (Roche Applied Science), etc., and the reaction conditions can be carried out according to the attached conditions.
  • microRNA As another method for detecting the function of microRNA, there can be mentioned a method for measuring the function depending on whether or not the translation of mRNA having the target base sequence of microRNA is suppressed.
  • MicroRNAs are known to suppress translation of mRNA containing the target nucleotide sequence in the 3 'terminal untranslated region (3' UTR) [Current Biology, 15, R458-R460 (20 05)]. Therefore, prepare a DNA in which the target base sequence for the single-stranded RNA to be measured is inserted into the 3 ′ UTR of an appropriate reporter gene expression vector, and conform to the expression vector. When it is introduced into a host cell and single-stranded RNA is expressed in that cell, the expression of the reporter gene is measured to detect whether it has a microRNA function! Can do.
  • the reporter gene expression vector may be any vector as long as it has a promoter upstream of the reporter gene and can express the reporter gene in the host cell.
  • any reporter gene can be used.
  • firefly luciferase gene renilla luciferase gene, chloramphenicol acetyltransferase gene, / 3-glucuronidase gene, The / 3—galactosidase gene, the / 3-lactamase gene, the equolin gene, the green 'fluorescent' protein gene and the DsRed fluorescent gene can be used.
  • Reporter gene expression vectors having such properties include, for example, psiCHE CK-1 (Promega), psiCHECK-2 (Promega), pGL3_Control (Promega), pGL4 (Promega), pRNAi-GL (Takara) Bio) and pCMV-DsRed-Express (CL ONTECH).
  • Single-stranded RNA can be expressed by the method described in 6 below.
  • RNA can be detected as follows. First, host cells are cultured in a multiwell plate or the like, and a reporter gene expression vector having a target sequence and single-stranded RNA are expressed. After that, the reporter activity is measured and the ability to detect the mechanism of the microphone RNA is measured by measuring the reporter activity when the single-stranded RNA is expressed compared to when not expressing the single-stranded RNA. it can.
  • the following describes a method for detecting the expression of microRNAs and their precursors expressed in mesenchymal stem cells.
  • Examples of methods for detecting the expression levels of microRNA and its precursor include (1) Northern hybridization, (2) Dot blot hybridization, and (3) In situ detection. Examples include hybridization, (4) quantitative PCR, (5) differential differential hybridization, (6) microarray, and (7) ribonuclease protection assay.
  • the Northern hybridization method is a method in which sample-derived RNA is separated by gel electrophoresis, Transfer to a support such as a nylon filter, prepare a probe with appropriate labeling based on the base sequence of the microRNA or microRNA precursor, and perform specific identification by washing, hybridization, and washing.
  • This is a method for detecting a bound band, specifically, for example, according to the method described in Science, 294, 853-858 (2001).
  • the labeled probe may be a radioisotope, biotin, digoxigenin, a fluorescent group, a chemiluminescent group, etc. by the method such as nick 'translation, random priming or phosphorylation at the 5' end. It can be prepared by incorporating it into DNA, RNA, or LNA having a sequence complementary to the base sequence of the RNA precursor. Since the binding amount of the labeled probe reflects the expression level of the microRNA or microRNA precursor, the expression level of the microRNA or microRNA precursor is quantified by quantifying the amount of bound labeled probe. can do. Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be carried out by the methods described in Molecular 3 ′ Cloning 3rd edition.
  • RNA extracted from tissues and cells is spot-fixed on a membrane in the form of spots, and labeled polynucleotides that serve as probes are hybridized with specific probes. This is a method for detecting RNA that hybridizes in a normal manner. A probe similar to Northern hybridization can be used. RNA preparation, RNA spots, hybridization, and RNA detection can be performed by the methods described in Molecular Cloning 3rd edition.
  • In situ hybridization uses a paraffin or cryostat section of tissue obtained from a living body or immobilized cells as a specimen, and performs a labeled probe, a hybridization, and a washing step. This is a method for examining the distribution and localization of the microRNA or microRNA precursor in tissues and cells [Metho ds in Enzymology, 254, 419 (1995)]. A probe similar to Northern hybridization can be used. Specifically, it is possible to detect microRNA according to the method described in Nature Method, 3, 27 (2006).
  • cDNA synthesized from a sample-derived RNA using a reverse transcription primer and a reverse transcriptase (hereinafter, the cDNA is referred to as a sample-derived cDNA) is used for measurement.
  • cDNA synthesis A random primer or a specific RT primer can be used as a reverse transcription primer for the preparation.
  • the specific RT primer refers to a primer having a sequence complementary to the base sequence corresponding to the microRNA or microRNA precursor used in the present invention and its surrounding genomic sequence.
  • RNA For example, after synthesizing a sample-derived cDNA, this is converted into a cage shape, and the nucleotide sequence lj corresponding to the cDNA or microRNA precursor and its surrounding genomic sequence, or the base corresponding to the reverse transcription primer. PCR is performed using a type-specific primer designed from the sequence to amplify a fragment of cDNA containing part or all of the microRNA or microRNA precursor, and the number of cycles until a certain amount is reached. The amount of nucleic acid containing the microRNA or microRNA precursor contained in the derived RNA is detected.
  • an appropriate region corresponding to the microRNA or microRNA precursor and its surrounding genomic sequence is selected, and the 5 ′ end 20-40 base sequence of the base sequence of the region is selected. It is possible to use DNA or LNA, and DNA or LNA consisting of a sequence complementary to 20 to 40 bases at the 3 ′ end. Specifically, it can be carried out according to the method described in Nucleic Acids Research, 32, e4 3 (2004).
  • a specific RT primer having a stem'loop structure can also be used as a reverse transcription primer for cDNA synthesis. Specifically, it can be performed using the method described in Nucleic Acid Research, 33, el79 (2005), or TaqMan MicroRNA Assays (Applied Systems).
  • a change in the amount of the microRNA or the microRNA precursor can be detected by performing hybridization on the derived cDNA and washing. Examples of such hybridization-based methods include a method using a differential 'hybridization [Trends Genet., 7, 314 (1991)] or a microarray [Genome Res., 6, 639 (1996)]. .
  • a nucleic acid having a nucleotide sequence corresponding to U6 RNA or the like is immobilized on a filter or substrate as an internal standard control, so that the difference in the amount of the nucleic acid of the present invention between the control sample and the target sample is eliminated. Can be detected accurately wear.
  • Labeled cDNA is synthesized using dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) that are labeled differently based on RNA from the control sample and sample, and can be used on a single filter or single substrate. By accurately hybridizing two labeled cDNAs, the microRNA or microRNA precursor can be accurately quantified.
  • microRNA can be detected using a microarray described in Proc. Natl. Acad. Sci. USA, 101, 9740-9744 (2004), Nucleic Acid Research, 32, el88 (2004), and the like. Specifically, it can be detected or quantified in the same manner as mirVana miRNA Bioarray (Ambion).
  • a promoter sequence such as a T7 promoter or SP6 promoter is bound to the 3 ′ end of the base sequence corresponding to the microRNA or microRNA precursor, or its surrounding genomic sequence, and labeled NTP (
  • a labeled antisense RNA is synthesized by an in vitro transcription system using a mixture of ATP, GTP, CTP and UTP) and RNA polymerase.
  • the labeled antisense RNA is bound to RNA derived from a sample to form an RNA-RNA oligonucleotide, and then digested with ribonuclease A that degrades only single-stranded RNA.
  • the digested product is subjected to gel electrophoresis, and an RNA fragment protected from digestion by forming an RNA-RNA hybrid is detected or quantified as the microRNA or microRNA precursor. Specifically, it can be detected or quantified using mirVana miRNA Detection Kit (Ambion).
  • Methods for detecting mutations in microRNA and microRNA precursors used in the present invention include normal microRNA and mutant microRNA, and normal microRNA precursor and mutant microRNA precursor. And a method for detecting a heteroduplex formed by hybridization.
  • Methods for detecting heteroduplex include (1) heteroduplex detection by polyacrylamide gel electrophoresis [Trends genet., 7, 5 (1991)], (2) —strand conformation Chillon polymorphism analysis [Genomics, 16, 325-332 (1993)], (3) Chemical cleavage or mismatches (CCM, num om Genetics (1996), ⁇ om Strachan and Andre w P Read, BIOS Scientific Publishers Limited], (4) Enzymatic cleavage method of mismatch [Nature Genetics, 9, 103-104 (1996)], (5) Denaturing gel electrophoresis [Mutat. Res., 288, 103 -112 (1993)].
  • the heteroduplex detection method by polyacrylamide gel electrophoresis is performed, for example, as follows. First, a fragment smaller than 200 bp using a primer designed based on the base sequence of the genome containing the base sequence of the DNA corresponding to the microRNA or microRNA precursor, using the sample-derived DNA or the sample-derived cDNA as a template. Amplify as. When heteroduplexes are formed, they are slower in mobility than homoduplexes without mutations and can be detected as extra bands. The degree of separation is better when special gels (Hydro-link, MDE, etc.) are used. Searching for fragments smaller than 200 bp can detect insertions, deletions, and most single base substitutions. Heteroduplex analysis is preferably performed on a single gel combined with the single-strand conformation analysis described below.
  • DNA bases corresponding to microRNAs or microRNA precursors using specimen-derived DNA or specimen-derived cDNA as templates.
  • DNA amplified as a fragment smaller than 200 bp is denatured and run in a native polyacrylamide gel.
  • the ability to label primers with isotopes or fluorescent dyes during DNA amplification, or the ability to detect amplified DNA as a band by silver-staining unlabeled amplification products is also run at the same time, a fragment with the difference can be detected from the difference in mobility.
  • the mismatch chemical cleavage method is designed based on the base sequence of the genome containing IJ, the base sequence of DNA corresponding to microRNA or microRNA precursor, using the sample-derived DNA or sample-derived cDNA as a template.
  • the DNA fragment amplified with the selected primer is hybridized with the labeled nucleic acid in which the microRNA or microRNA precursor is incorporated with an isotope or fluorescent label, and treated with osmium tetroxide. Mutation can be detected by cleaving one strand.
  • CCM is one of the most sensitive detection methods and can be applied to specimens of kilobase length.
  • mismatch By combining RNaseA with an enzyme involved in mismatch repair in cells such as T4 phage resol base and endonuclease VII instead of osmium tetroxide, The mismatch can also be cleaved enzymatically.
  • DGGE denaturing gradient gel electrophoresis
  • genomic bases that contain DNA sequences corresponding to microRNA or microRNA precursors using specimen-derived DNA or specimen-derived cDNA as a template.
  • DNA fragments amplified with primers designed based on the sequence are electrophoresed using a gel with a chemical denaturant concentration gradient or temperature gradient.
  • the amplified DNA fragment moves to the position where it is denatured into a single strand in the gel and does not move after denaturation.
  • the presence of the mutation can be detected because the movement of the amplified DNA in the gel differs in the presence and absence of the mutation.
  • attach a poly (G: C) terminal to each primer! /.
  • mutations in microRNA or microRNA precursors can be detected by directly determining and analyzing the base sequence of specimen-derived DNA or specimen-derived cDNA.
  • a microRNA or derivative thereof, a microRNA precursor or derivative thereof, or a double-stranded nucleic acid used in the present invention is introduced into a cell and the microRNA or derivative thereof, a microRNA precursor or derivative thereof, or a double-stranded nucleic acid.
  • By transcribing the double-stranded nucleic acid it can be expressed by using a vector that is biosynthesized. Specifically, based on the base sequence of the microRNA or derivative thereof, the microRNA precursor or derivative thereof, or a double-stranded nucleic acid, or the genomic base sequence containing the base sequence, DNA containing a hairpin portion.
  • a fragment is prepared, inserted into a promoter in an expression vector to construct an expression plasmid, and then the expression plasmid is introduced into a host cell suitable for the expression vector to thereby produce the microRNA or a derivative thereof.
  • MicroRNA precursors or derivatives thereof, or double stranded nucleic acids can be expressed.
  • the expression vector can replicate autonomously in a host cell or can be integrated into a chromosome, and can be used for microRNA or a derivative thereof, a microRNA precursor or a derivative thereof, or a double-stranded nucleic acid used in the present invention. Those containing a promoter at a position where a gene containing the base sequence can be transcribed are used. Any promoter can be used as long as it can be expressed in the host cell.For example, RNA polymerase Il (p ol iii) type promoters and RNA polymerase III (pol III) type promoters that are transcription systems of U6 RNA and HI RNA.
  • Examples of the pol II promoter include a promoter of cytomegalovirus (human CMV) IE (immediate early) gene, SV40 early promoter, and the like.
  • Examples of expression vectors using them include pCDNA6.2-GW / miR (Invitrogen) and pSilencer 4.1_CMV (Ambion).
  • Examples of pol III promoters include U6 RNA, HI RNA, and tRNA promoters.
  • Examples of expression vectors using them include pSI Nsi-hHl DNA (Takara Bio), pSINsi_hU6 DNA (Takara Bio), pENTR / U 6 (Invitrogen) and the like.
  • a recombinant viral vector obtained by inserting a microRNA or a derivative thereof used in the present invention, a microRNA precursor or a derivative thereof, or a gene containing a base sequence of a double-stranded nucleic acid downstream of a promoter in a viral vector. And the vector is introduced into a packaging cell to produce a recombinant virus, and the base sequence of the microRNA or derivative thereof, microRNA precursor or derivative thereof, or double-stranded nucleic acid used in the present invention A gene containing can also be expressed.
  • a cell / caging cell may be any cell that can replenish the deficient protein of a recombinant viral vector deficient in any of the genes encoding the proteins required for viral packaging.
  • HEK293 cells derived from human kidney, mouse fibroblasts MH3T3, etc. can be used.
  • Proteins supplemented by packaging cells include mouse retrovirus-derived gag, pol, env, etc. for retroviral vectors, and HIV virus-derived gag, pol, env, vpr for lentiviral vectors.
  • Nude protein in the case of adenovirus vectors, proteins such as ⁇ ⁇ ⁇ , ⁇ derived from adenovirus, and R in the case of adeno-associated virus vectors ( ⁇ 5, ⁇ 19, p40) , Proteins such as Vp (Cap) can be used.
  • the microRNA or derivative thereof, microRNA precursor or derivative thereof, or double-stranded nucleic acid used in the present invention can also be directly introduced into a cell without using a vector.
  • these chimeric molecules or derivatives of these nucleic acids may be used as nucleic acids for this method. it can.
  • Pre-miRTM miRNA Precursor Molecules Ambion
  • miRI DIAN microRNA Mimics GE Healthcare
  • microRNA When microRNA is expressed, any method may be used as long as microRNA can finally be produced in the cell, but preferably (1) single-stranded RNA that is a microRNA precursor, (2 Examples include a method of introducing a double-stranded RNA consisting of a microRNA and a complementary strand of microRNA, or (3) a double-stranded RNA that assumes a state after the microRNA is cut into Dicers.
  • the microRNA used in the present invention is expressed in the same manner as miCENTURY OX Precursor (B-Bridge), miCENTURY OX siMature (B-Bridge), and miCENTURY OX miNatural (B-Bridge). be able to.
  • microRNA or microRNA precursor used in the present invention is an antisense technology [Bioscience and Industry, 50, 322 (1992), Chemistry, 46, 681 (1991), Biotechnology, 9, 358 (1992). , Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992), Cell Engineering, 16, 6463 (1997)], Triple Helix Technology [Trends in Biot echnology, 10, 132 (1992) )], Rehoc technology [Current Opinion in Chemical Biology, 3, 274 (1999), FEMS Microbiology Reviews, 23, 257 (1999), Frontiers in Bioscience, 4, D497 (1999), Chemistry & Biology, 6, R33 ( 1999), Nucleic Acids Research, 26, 5 237 (1998), Trends In Biotechnology, 16, 438 (1998)], Decoy DNA method [Nippon Rinsho-Japanese Journal of Clinical Medicine, 56, 563 (1998), Circulation Research, 82, 1023 (1998), Experimental Nephrology, 5, 429 (1997), Nippon Rinsho-Japanese Journal
  • Antisense refers to a nucleic acid having a base sequence complementary to a base sequence of a certain target nucleic acid that can specifically hybridize to the base sequence to suppress the expression of the target nucleic acid.
  • these chimeric molecules or derivatives of the nucleic acids can also be used as nucleic acids for antisense. Specifically, Natu By following the method described in re, 432, 226 (2004), etc., it is possible to produce an antisense and suppress the expression.
  • anti-miRTM miRNA Inhibitors manufactured by Amb ion
  • miRI DIAN microRNA Inhibitors manufactured by GE Healthcare
  • the siRNA is a short double-stranded RNA containing a base sequence of a certain target nucleic acid, and can suppress the expression of the target nucleic acid by RNA interference (RN Ai).
  • the siRNA IJ can be appropriately designed based on the conditions of the literature [Genes Dev., 13, 3191 (1999)] based on the target nucleotide sequence.
  • a siRNA can be prepared by synthesizing and annealing two RNAs each having a 19-base sequence selected and a complementary sequence each having a TT added at the 3 'end, and annealing.
  • siRNA expression vector such as pSilencer 1.0-U6 (Ambion) or pSUPER (01igoEngine)
  • the expression of the gene can be suppressed. It is possible to create vectors that express siRNA.
  • microRNA expressed in mesenchymal stem cells or the microRNA precursor using microRNA expressed in mesenchymal stem cells or antisense or siRNA specific to the microRNA precursor can be suppressed. That is, by applying antisense DNA or siRNA specific to the microRNA or the microRNA precursor, the expression of the microRNA is suppressed, and the action of microRNA or microRNA precursor in mesenchymal stem cells Can be controlled.
  • microRNA or its precursor expressed in mesenchymal stem cells by administering antisense or siRNA specific to the microRNA or its precursor to the patient, By controlling the function of mesenchymal stem cells, it is possible to treat diseases caused by the abnormal expression. That is, antisense or siRNA specific to the microRNA or its precursor is useful as a therapeutic agent for diseases caused by abnormal mesenchymal stem cells.
  • antisense or siRNA specific to the microRNA or its precursor is used as the above therapeutic agent, antisense or siRNA alone, or a retrovirus vector, adenovirus vector, adeno-associated virus vector, etc.
  • Suitable vector After insertion, it can be administered as a pharmaceutical preparation according to conventional methods described in 10 and 11 below.
  • microRNA is the mRNA of the target gene. Any method that uses the activity of suppressing translation to suppress the expression of a gene having a target base sequence can be used. For example, by increasing the intracellular level of microRNA, The ability to suppress the translation of the target gene mRNA and to suppress the expression of the target gene can be improved.
  • the microRNA or microRNA precursor can be expressed in cells by the method described in 6 above. Examples of the target gene of the microRNA consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 93 include the gene groups shown in Table 1 above, for example.
  • the method of screening for an agent for inhibiting the proliferation and / or differentiation of mesenchymal stem cells of the present invention includes (A) promoting the expression of microRNA or microRNA precursor used in the present invention, and (B) presenting the present invention. Inhibiting the expression of the target gene of the microRNA to be used can be used as an indicator. Further, the screening method for the mesenchymal stem cell proliferation and / or differentiation promoting agent of the present invention includes (C) suppressing the expression of microRNA or microRNA precursor used in the present invention, (D) Examples of the method that can be used to promote the expression of the target gene of the microRNA used in the present invention can be mentioned.
  • a base sequence to be screened is selected and selected using a cell that expresses a nucleic acid having the base sequence.
  • a substance that promotes or suppresses the expression of the microRNA or the microRNA precursor can be screened.
  • the cells expressing nucleic acids having the base sequences of microRNA and microRNA precursor used for screening include mesenchymal stem cells as well as vectors expressing nucleic acids having the base sequences as described in 6 above. It is also possible to use a transformed cell obtained by introducing the DNA into a host cell such as an animal cell or yeast, or a cell into which a nucleic acid having the base sequence is directly introduced without using a vector.
  • Specific screening methods include: (a) a method that uses changes in the expression level of microRNAs or microRNA precursors targeted for screening as an index, and (b) a target gene for microRNAs that is targeted for screening. A method using as an index the change in mRNA expression level.
  • a test substance is brought into contact with a cell that expresses a nucleic acid having the nucleotide sequence, and a substance that suppresses or promotes the proliferation and / or differentiation of mesenchymal stem cells is used as an indicator of the change in the expression level of the selected nucleic acid. obtain.
  • the amount of nucleic acid expressed can be detected by the method S described in 3 above.
  • DNA in which the target sequence for the microRNA used in the present invention is inserted into the 3 ′ UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector.
  • a test substance is contacted, and a substance that suppresses proliferation and / or differentiation of mesenchymal stem cells is obtained using the change in the expression level of the reporter gene as an index.
  • the selection of the target base sequence can be performed by the method described in the above 8.
  • the target gene of the microRNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 93 is, for example, as described above.
  • the gene group shown in Table 1 can be exemplified.
  • nucleic acid-containing diagnostics and therapeutics used in the present invention controls the expression of the target gene of the microRNA or microRNA precursor or the microRNA Thus, it can be used as a therapeutic agent for diseases caused by abnormal proliferation or differentiation of mesenchymal stem cells.
  • it can be used as a diagnostic agent for diseases caused by abnormal proliferation or differentiation of leaf stem cells.
  • siRNA against the target gene of the microRNA can be used as a therapeutic agent for diseases caused by abnormalities in proliferation and / or differentiation of mesenchymal stem cells by controlling the mRNA expression of the target gene of the microRNA.
  • diseases caused by abnormal proliferation or differentiation of mesenchymal stem cells and the like include cancer, osteogenesis imperfecta, cartilage imperfecta and diabetes.
  • a therapeutic agent containing, as an active ingredient, a nucleic acid used in the present invention, a nucleic acid containing a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or siRNA against the target gene of the microRNA can be administered alone, but is usually mixed with one or more pharmacologically acceptable carriers and prepared by any method well known in the pharmaceutical arts. It is desirable to administer as
  • the route of administration is preferably oral, for which it is desirable to use the most effective treatment, or parenteral, such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous. It is possible to increase the dosage, preferably intravenous administration.
  • Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P-hydroxybenzoate Preservatives such as acid esters, flavors such as stove leaf flavor and peppermint can be used as additives.
  • Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, It can be produced by using a binder such as bull alcohol, hydroxypropyl cellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • a binder such as bull alcohol, hydroxypropyl cellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • Suitable formulations for parenteral administration include injections, suppositories, sprays and the like.
  • An injection is prepared using a carrier comprising a salt solution, a glucose solution or a mixture of both.
  • Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa and that facilitates absorption by dispersing the active ingredient as fine particles.
  • the carrier include lactose and glycerin.
  • preparations such as aerosols and dry powders are possible depending on the nature of the carrier used.
  • the components exemplified as additives for oral preparations can also be added.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc.
  • the usual adult dose is 10 Hg / kg to 20 mg / kg per day.
  • a therapeutic agent containing, as an active ingredient, a nucleic acid used in the present invention, a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or siRNA against a target gene of microRNA A nucleic acid used in the present invention, a nucleic acid containing a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or a vector expressing siRNA against a target gene of microRNA and a base used for a nucleic acid therapeutic agent It can also be produced by blending [Nature Genet., 8, 42 (1994)].
  • the base used in the nucleic acid therapeutic agent may be any base that is usually used for injections, and is a salt solution such as distilled water, sodium chloride, or a mixture of sodium chloride and an inorganic salt. , Mannitol, ratatoose, dextran, glucose and the like, amino acid solutions such as glycine and arginine, organic acid solutions or mixed solutions of a salt solution and a glucose solution, and the like.
  • these bases are mixed with an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil and die oil, or an auxiliary agent such as a lecithin or a surfactant such as a nonionic surfactant.
  • An injection may be prepared as a suspension or dispersion. These injections can also be prepared as preparations for dissolution upon use by operations such as pulverization and freeze-drying.
  • the therapeutic agent of the present invention can be used for treatment as it is in the case of a liquid just before the treatment, and in the case of an individual, it can be dissolved in the above sterilized base as necessary.
  • nucleic acid used in the present invention a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence consisting of SEQ ID NOs: 1 to 201, or a vector containing siRNA against a target gene of microRNA is the above 6
  • the prepared recombinant virus vector include virus vectors, and more specifically, a retrovirus vector and a lentivirus vector.
  • a nucleic acid used in the present invention a nucleic acid containing a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or a siRNA against a microRNA target gene is specific to an adenovirus .
  • a viral vector can be prepared by preparing a complex by combining with a typical polylysine-conjugated antibody and binding the resulting complex to an adenoviral vector. The viral vector stably reaches the target cell, is taken up into the cell by endosomes, and is degraded in the cell to efficiently express the nucleic acid.
  • microRNAs used in the present invention can be obtained using the Sendai virus. Or, you can use S to produce a Sendai virus that incorporates a microRNA precursor.
  • nucleic acid used in the present invention a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or siRNA for a target gene of microRNA is a non-viral nucleus. It can also be transferred by the acid transfer method. For example, calcium phosphate coprecipitation method [Virology, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980); Proc.Natl.Acad.Sci.
  • Ribosome-mediated membrane fusion-mediated transfer is a method in which a ribosome preparation is directly administered to a target tissue so that it is complementary to the nucleic acid used in the present invention, the base sequence consisting of SEQ ID NOs: 1 to 201.
  • a nucleic acid containing a base sequence or siRNA for a target gene of microRNA can be taken up locally and expressed in the tissue [Hum. Gene Ther., 3, 399 (1992)].
  • Direct DNA uptake techniques are preferred for targeting DNA directly to the lesion.
  • Receptor-mediated DNA transfer is performed, for example, by binding DNA (typically in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine.
  • the ligand is selected based on the presence of the corresponding ligand receptor on the cell surface of the cell or tissue of interest.
  • the ligand-DNA complex can be injected directly into the blood vessel, if desired, and can be directed to a target tissue where receptor binding and internalization of the DNA-protein complex occurs.
  • adenovirus can be co-infected to disrupt endosomal function. 11.Differentiation and evaluation method from ⁇ ⁇ ⁇ to osteoblast ⁇
  • the nucleic acid used in the present invention or siRNA for the target gene of microRNA is introduced into mesenchymal stem cells and cultured. . Analyze genes or proteins that increase in expression as they differentiate into osteoblasts and compare to negative controls
  • any method capable of inducing differentiation from mesenchymal stem cells to osteoblasts! / any method capable of inducing differentiation from mesenchymal stem cells to osteoblasts! /, Any method may be used! /
  • the method described in Science, 284, 143-147 (1999) can be mentioned.
  • mesenchymal stem cells can be differentiated into osteoblasts.
  • Quantitative analysis of genes whose expression varies with differentiation into osteoblasts includes RT-P, R, reverse transcription—polymerase chain reaction, nozano, yipryta, and dot blot hybridiy. Examples include a method using a seed or DNA microarray.
  • Quantitative analysis methods for proteins whose expression varies with osteoblast differentiation include Western blot analysis, immunohistochemical staining using antibodies that react specifically with the protein, and ELI SA. .
  • Genes or proteins whose expression increases with differentiation into osteoblasts include type I collagen, osteocalcin, osteonetatin, osteopontin, bone sialoprotein, Runx2 (runt-related gene 2) Anoleka diphosphatase T (ALP) and the like.
  • Examples of methods for evaluating the degree of differentiation into osteoblasts include a method of staining cells using the ALP enzyme activity in the osteoblast, or a method of measuring ALP enzyme activity.
  • a more specific method of the cell staining is to couple the alcohol portion of the phosphate ester of the substrate hydrolyzed by ALP enzyme in osteoblasts with a diazonium salt,
  • a method of precipitating with an azo dye on the enzyme active site can be mentioned.
  • the substrate for example, naphthonor AS-MX phosphoric acid can be mentioned, and as the azo dye, for example, first violet blue can be mentioned.
  • a kit containing these may be used, such as leukocyte alkaline phosphatase (manufactured by Sigma).
  • a kit for measuring ALP enzyme activity for example, alkaline phospha B-test coco (manufactured by Wako Pure Chemical Industries, Ltd.) may be used.
  • the differentiation into osteoblasts can also be confirmed by detecting the calcification component produced by the osteoblasts.
  • the method for detecting the calcified component include staining methods such as von ossa staining and alizarin red staining.
  • Von Kossa staining is a method of detecting calcium phosphate, which is a calcifying component, using silver nitrate. Specifically, the cells fixed with paraffin or the like are reacted with 1 to 5% silver nitrate aqueous solution and exposed to light, and the portion where the black calcium phosphate is present is quantified, for example, the bone area is measured by measuring the colored area. The degree of differentiation into blast cells can be evaluated.
  • Alizarin red staining is a method utilizing the fact that alizarin red S shows a specific binding to calcium and forms a rake. Specifically, by reacting 0.01 to 5% alizarin red S solution with cells fixed with paraffin or the like, the colored portion of reddish purple to orange red is quantified, for example, by measuring the colored area. Ability to evaluate the degree of differentiation into osteoblasts.
  • hMSC Human mesenchymal stem cells
  • Cambrex Cambrex
  • IMDM medium Invitrogen
  • FBS urine fetal serum
  • RNA was extracted from hMSC in culture using TRIZOL reagent (Invitrogen) according to the method attached to the product.
  • microRNAs expressed in mesenchymal stem cells include, for example, SEQ ID NO: 1 (hsa-miR-24), SEQ ID NO: 2 (hsa_miR-100), SEQ ID NO: 3 (hsa-miR-145). ), SEQ ID NO: 4 (hsa-miR-210) and SEQ ID NO: 5 (hsa-miR-422b).
  • microRNA precursor obtained in Example 1 was introduced into hMSC, cultured under conditions for inducing differentiation into osteoblasts, and the influence of the microRNA precursor was examined.
  • hMSCs were seeded in a 24-well plate so that there were 6 ⁇ 2 ⁇ 10 3 per well, and cultured in a DM medium containing 20% FBS.
  • the microRNA precursor was introduced into the hMSC using a lipofection method, specifically, Lipofectamine 2000 (manufactured by Invitrogen) to a final concentration force of 3 ⁇ 40 nM.
  • a lipofection method specifically, Lipofectamine 2000 (manufactured by Invitrogen) to a final concentration force of 3 ⁇ 40 nM.
  • Ambion's Pre-miR TM miRNA Precursor Molecules was used, and the lipofusion method was in accordance with the method attached to the product.
  • the medium was induced to induce osteoblast differentiation [in IMDM medium containing 20% FBS, 0.1 ⁇ mol / L dexamethasone, 50 ⁇ mol / L ascorbic acid- 2 phosphoric acid (manufactured by Sigma), 10 mmol / L / 3-glyce mouth phosphate (manufactured by Sigma)], and the osteoblast differentiation induction medium was replaced once every three days to continue the culture.
  • IMDM medium containing 20% FBS, 0.1 ⁇ mol / L dexamethasone, 50 ⁇ mol / L ascorbic acid- 2 phosphoric acid (manufactured by Sigma), 10 mmol / L / 3-glyce mouth phosphate (manufactured by Sigma)]
  • the osteoblast differentiation induction medium was replaced once every three days to continue the culture.
  • the cell morphology was observed under a phase contrast microscope (Nikon), and further, alkaline phosphatase staining was performed to detect osteoblasts
  • the cells were washed once with phosphate-buffered saline (PBS) (manufactured by Invitrogen) and fixed with fixative (10% formalin / PBS) for 5 minutes. After washing with distilled water, the mixture was reacted with a mixed solution of Naphthol AS-MX phosphate (manufactured by Sigma) and Fast Violet B solution (manufactured by Sigma) for 30 minutes to perform an alkaline phosphatase reaction. Further, it was washed with distilled water, stained red under a phase contrast microscope! /, And the osteoblasts were observed and photographed with a digital camera (Nikon).
  • PBS phosphate-buffered saline
  • hMSCs with hsa-miR-24, hsa-miR-100, hsa-miR-145, hsa-miR-210, and hsa-miR-422b precursors were introduced without microRNA precursors. It was found that the number of positive cells stained with alkaline phosphatase was smaller than that of hMSC. Since the microRNA precursor is converted into microRNA in the cell, the microRNA used in this example has an activity to suppress proliferation and an activity to suppress differentiation into osteoblasts against hMSC. Kotawa power.
  • hMSC is cultured in the above osteoblast differentiation medium, 1 day, 3 days, 5 days after the start of culture,
  • mirVana miRNA Isolation Kit (Ambion) was used and the method attached to the product was followed.
  • the expression level was calculated as the A Ct value after obtaining the number of cycles (Ct value) when the signal intensity reached 1000 and then subtracting the above value from the negative control Ct value.
  • Negative control used the Ct value of PCR using sterilized water.
  • the expression level of hMSC before differentiation induction was 1.0, and the relative expression level of each was calculated. As a result, as shown in Fig. 1, the expression level of any microRNA at the initial stage of osteoblast differentiation was There has been no significant increase in power.
  • Example 2 Searched for hsa-miR-145 target gene candidates that showed hMSC proliferation and differentiation suppression activity in Example 2, and introduced siRNAs of ADD3 gene and CBFB gene into hMSC to suppress the expression of the gene The process of hMSC differentiation into osteoblasts under the conditions was investigated. In the same manner as in Example 2, hMSCs were cultured in an osteoblast differentiation induction medium.
  • siRNAs that suppress the expression of the ADD3 gene (SEQ ID NO: 206)
  • the siRNA targeting the nucleotide sequence represented by SEQ ID NO: 202 and the siRNA targeting the nucleotide sequence represented by SEQ ID NO: 203 are chemically synthesized. did.
  • siRNAs that suppress the expression of the CBFB gene (SEQ ID NO: 207)
  • siRNA targeting the nucleotide sequence represented by SEQ ID NO: 204 and siRNA targeting the nucleotide sequence represented by SEQ ID NO: 205 were chemically synthesized.
  • the chemically synthesized double-stranded RNA was introduced into hMSC by the lipofusion method.
  • the hsa-miR-145 precursor that showed inhibitory activity against hMSC proliferation and differentiation in Example 4 was introduced into hMSC, and changes in the amount of protein encoded by the target gene were examined.
  • Lipofectam with Ambion hsa-miR-145 precursor to a final concentration of 2nM or 20nM It was introduced into 1.2 x15 5 hMSCs previously seeded with ine2000. Three days after introduction, the cells were dissolved in Lysis buffer [50 mM Tris—HCL (pH 7.4), 150 mM NaCl, ImM EDTA, 1% Nonident P-40, 1% Protease Inhibitor Cocktail Set III (Calbiochem)]. It was collected. After separation by SDS-PAGE by a conventional method, the amounts of ADD3 and CBFB proteins were detected and quantified by Western blotting. Anti-ADD3 antibody (manufactured by SantaCruz) was used for detection of ADD3, and anti-CBFB antibody (manufactured by Abeam) was used for detection of CBFB.
  • the hMSCs that forcibly expressed the hsa-miR-145 precursor had decreased amounts of Addcin3 and CBFB proteins compared to the negative control hMSC, so the ADD3 and CBFB genes were the target genes of hsa_m iR-145. It was shown that expression is regulated.
  • a nucleic acid such as microRNA or a derivative thereof of the present invention is effective in suppressing the proliferation and / or differentiation of mesenchymal stem cells, suppressing the expression of a target gene of microRNA, and abnormally growing and / or differentiating mesenchymal stem cells. It is useful for diagnosis and treatment of diseases caused by it.

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Abstract

Disclosed are: microRNA or a derivative thereof; a microRNA precursor or a derivative thereof: a double-stranded nucleic acid composed of the microRNA or derivative thereof and a nucleic acid comprising a nucleotide sequence complementary to a nucleotide sequence for the microRNA or derivative thereof; an inhibitor of the growth and/or differentiation of a mesenchymal stem cell, which comprises a double-stranded nucleic acid composed of the microRNA or derivative thereof and a nucleic acid capable of hybridizing with the microRNA or derivative thereof under stringent conditions; an inhibitor of the expression of a microRNA target gene; use of the inhibitor; and a diagnostic or therapeutic agent for a disease induced by the abnormality in the growth and/or differentiation of a mesenchymal stem cell.

Description

明 細 書  Specification

間葉系幹細胞の増殖および/または分化制御剤  Mesenchymal stem cell proliferation and / or differentiation regulator

技術分野  Technical field

[0001] 本発明は、間葉系幹細胞の増殖および/または分化抑制剤、核酸の 1種であるマ イク口 RNAの標的遺伝子の発現抑制剤、間葉系幹細胞の増殖および/または分化 抑制方法、マイクロ RNAの標的遺伝子の発現抑制方法、間葉系幹細胞の増殖およ び/または分化抑制剤のスクリーニング方法、間葉系幹細胞の増殖および/または 分化促進剤、間葉系幹細胞の増殖および/または分化促進方法、間葉系幹細胞の 増殖および/または分化の異常に起因する疾患の診断薬、間葉系幹細胞の増殖お よび/または分化の異常に起因する疾患の治療薬に関する。  [0001] The present invention relates to an inhibitor of proliferation and / or differentiation of mesenchymal stem cells, an inhibitor of expression of a target gene of a mouthpiece RNA that is one of nucleic acids, and a method of inhibiting proliferation and / or differentiation of mesenchymal stem cells. , MicroRNA target gene expression suppression method, mesenchymal stem cell proliferation and / or differentiation inhibitor screening method, mesenchymal stem cell proliferation and / or differentiation promoter, mesenchymal stem cell proliferation and / or The present invention also relates to a method for promoting differentiation, a diagnostic agent for a disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells, and a therapeutic agent for a disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells.

背景技術  Background art

[0002] 核酸の 1種であるマイクロ RNA(miRNA)は、蛋白質に翻訳されない約 22ヌクレオチド の小さな非コード一本鎖 RNAであり、ヒトを含む生物に多数存在することが確認され ている（非特許文献 1、 2)。  [0002] MicroRNA (miRNA), a kind of nucleic acid, is a small non-coding single-stranded RNA of about 22 nucleotides that is not translated into protein, and has been confirmed to exist in many organisms including humans (non-native). Patent Documents 1 and 2).

マイクロ RNAは、単一又はクラスター化されたマイクロ RNA前駆体に転写される遺伝 子から生成される。すなわち、まず、遺伝子から一次転写産物である primary-microR NA (pri-miRNA)が転写され、次いで、 pri-miRNAから成熟型マイクロ RNAへの段階的 プロセシングにお!/、て、 pri-miRNAから特徴的なヘアピン構造を有する約 70塩基の pr ecursor-microRNA (pre-miRNA)が生成される。さらに、 Dicer介在によるプロセシング により pre-miRNAから成熟型マイクロ RNAが生成される（非特許文献 3)。  MicroRNAs are generated from genes that are transcribed into single or clustered microRNA precursors. That is, first, primary-microRNA (pri-miRNA), a primary transcript, is transcribed from the gene, and then for stepwise processing from pri-miRNA to mature microRNA! /, From pri-miRNA Approximately 70 base pr ecursor-microRNA (pre-miRNA) having a characteristic hairpin structure is produced. Furthermore, mature microRNA is produced from pre-miRNA by Dicer-mediated processing (Non-patent Document 3).

[0003] 成熟型マイクロ RNAは、標的となる mRNAに相補的に結合して mRNAの翻訳を抑制 する、あるいは mRNAを分解することにより、遺伝子発現の転写後制御に関与してい ると考えられている。 2006年 5月現在、マイクロ RNAのデータベース miRBase (http：〃 microrna.sanger.ac.uk/)には、ヒトで 455種、全生物種で 3685種のマイクロ RNAが登 録されている。ヒトを含む哺乳類で発現するマイクロ RNAの中で、その生理的機能に 関してわかっているものは、血球系分化に関与する miR-181 (非特許文献 4)やインシ ュリン分泌に関与する miR-375 (非特許文献 5)、癌細胞の増殖を抑制する miR_143、 miR-145 (非特許文献 11)、癌細胞の増殖を促進する miR-372、 miR_373 (非特許文 献 12)など一部のみであり、多くはその生理的活性が未解明である。ただし、線虫や ショウジヨウバエを用いた研究からマイクロ RNAが生物の発生、分化に様々な重要な 役割を果たしていることが明ら力、となってきており、ヒト疾患との関係においても特に 癌との深い関係を示唆する報告が出てきている (非特許文献 6)。 [0003] Mature microRNAs are thought to be involved in post-transcriptional regulation of gene expression by binding complementarily to the target mRNA and suppressing translation of the mRNA, or by degrading the mRNA. Yes. As of May 2006, the microRNA database miRBase (http: microrna.sanger.ac.uk/) contains 455 human and 3685 microRNAs in all living species. Among the microRNAs expressed in mammals including humans, those that are known for their physiological functions include miR-181 (Non-patent Document 4) involved in blood cell differentiation and miR- involved in secretion of insulin. 375 (Non-Patent Document 5), miR_143 that suppresses the growth of cancer cells, miR-145 (Non-patent document 11), miR-372 that promotes cancer cell growth, miR_373 (Non-patent document 12), etc. are only some of them, and many of them have unclear physiological activity. However, studies using nematodes and Drosophila have revealed that microRNAs play various important roles in the development and differentiation of organisms, especially in relation to human diseases. There have been reports suggesting a deep relationship with cancer (Non-patent Document 6).

[0004] マイクロ RNAの同定には、細胞から低分子 RNAクローニングする方法や、ゲノム配 列情報からバイオインフォマティクスを用いる方法などがある。 miRBaseにマイクロ RN Aとして登録されるためには、発現に関する情報と生合成及び構造に関する情報の 両方が必要であり、ゲノム配列情報から構造の予測だけではマイクロ RNAとして認め られていない (非特許文献 7)。  [0004] For identification of microRNA, there are a method of cloning small RNA from a cell, a method of using bioinformatics from genome sequence information, and the like. In order to be registered as a microRNA in miRBase, both information related to expression and information related to biosynthesis and structure are required, and it is not recognized as a microRNA simply by predicting the structure from genomic sequence information (Non-patent Documents). 7).

[0005] 間葉系幹細胞は、哺乳類の骨髄、脂肪組織、臍帯血等に存在し、脂肪細胞、軟骨 細胞、骨細胞等に分化する多能性の幹細胞として知られている。間葉系幹細胞は、 その分化多能性の故に、骨、軟骨、腱、筋肉、脂肪、歯周組織など、多くの組織の再 生医療のための移植材料として注目されて!/、る（非特許文献 8)。  [0005] Mesenchymal stem cells are known as pluripotent stem cells that are present in mammalian bone marrow, adipose tissue, umbilical cord blood, and the like and differentiate into adipocytes, chondrocytes, bone cells, and the like. Because of their pluripotency, mesenchymal stem cells are attracting attention as a transplant material for regenerative medicine of many tissues such as bone, cartilage, tendon, muscle, fat, and periodontal tissue! Non-patent document 8).

間葉系幹細胞は、薬剤やサイト力イン等の添加により in vitroで特定の細胞へ分化 させること力 Sでき、例えば、脂肪細胞へは 1-メチル -3-イソブチルキサンチン、デキサ メタゾン、インスリンおよびインドメタシンを作用させることにより、骨芽細胞へはデキサ メタゾン、 β—グリセロールフォスフェイト、およびァスコルビン酸を作用させることによ り、誘導できる（非特許文献 9)。しかし、これらの分化の過程における分子的なメカ二 ズムに関しての詳細は不明である。遺伝子ノックアウトマウスや分化段階での遺伝子 発現解析の結果から、脂肪細胞へは PPAR yおよび C/EBPファミリーが、骨芽細胞分 化時には Cbfal/Runx2および Osterkなどの遺伝子発現が関与していることが知られ ている（非特許文献 10)が、これらの遺伝子群のみで間葉系幹細胞からの分化メカ 二ズムを説明することはできず、人為的に分化及び増殖を制御するには至っていな い。また、間葉系幹細胞の分化及び増殖に作用するマイクロ RNAは知られていない。 非特許文献 1：「サイエンス (Science) J、 2001年、 294巻， p.853-858  Mesenchymal stem cells can be differentiated into specific cells in vitro by the addition of drugs, cytodynamic force, etc. For example, 1-methyl-3-isobutylxanthine, dexamethasone, insulin and indomethacin can be differentiated into adipocytes. Can be induced by causing dexamethasone, β-glycerol phosphate, and ascorbic acid to act on osteoblasts (Non-patent Document 9). However, details regarding the molecular mechanisms in these differentiation processes are unknown. Based on the results of gene expression analysis in gene knockout mice and differentiation stages, it is clear that PPAR y and C / EBP families are involved in adipocytes, and that gene expression such as Cbfal / Runx2 and Osterk is involved in osteoblast differentiation. Although it is known (Non-patent Document 10), these gene groups alone cannot explain the differentiation mechanism from mesenchymal stem cells, and have not yet been able to artificially control differentiation and proliferation. Yes. In addition, microRNAs that affect the differentiation and proliferation of mesenchymal stem cells are not known. Non-Patent Document 1: “Science J, 2001, 294, p.853-858

非特許文献 2 :「セル（Cell)」、 2003年、 113巻， p.673-676  Non-Patent Document 2: “Cell”, 2003, 113, 673-676

非特許文献 3 :「ネイチヤー レビュー ジェネティックス（Nature Reviews Genetics)」，2 004年、 5巻， .522-531 Non-Patent Document 3: "Nature Reviews Genetics", 2 004, volume 5, .522-531

非特許文献 4 :「サイエンス（Science)」， 2004年、 303巻， .83-86  Non-Patent Document 4: "Science", 2004, 303, .83-86

非特許文献 5 :「ネイチヤー（Nature)」， 2004年、 432巻， 226-230  Non-Patent Document 5: “Nature”, 2004, 432, 226-230

非特許文献 6 :「ネイチヤー レビュー キャンサー（Nature Reviews Cancer)」， 2006 年、 6巻， 259-269  Non-Patent Document 6: “Nature Reviews Cancer”, 2006, Vol. 6, 259-269

非特許文献 7 :「アールェヌエイ（RNA)」， 2003年、 9巻， 277-279  Non-Patent Document 7: “NA”, 2003, Vol. 9, 277-279

非特許文献 8 :「遺伝子医学」、 2000年、 4巻、 p. 58 - 61  Non-Patent Document 8: “Gene Medicine”, 2000, vol. 4, p. 58-61

非特許文献 9 :「サイエンス（Science)」、 1999年、 284巻、 ρ· 143-147  Non-Patent Document 9: “Science”, 1999, 284, ρ · 143-147

非特許文献 10 :「実験医学」、 2002年、 20巻、 p. 2459 - 2464  Non-Patent Document 10: "Experimental Medicine", 2002, 20, p. 2459-2464

非特許文献 11：「オンコロジ一レポート (Oncology Report)]、 2006年、 16巻、 p.845-85 Non-Patent Document 11: “Oncology Report”, 2006, 16, 845-85

0 0

非特許文献 12 :「セル（Cell)」、 2006年、 124巻、 p. 1169-1181  Non-Patent Document 12: “Cell”, 2006, 124, p. 1169-1181

発明の開示  Disclosure of the invention

発明が解決しょうとする課題  Problems to be solved by the invention

[0006] ヒトの種々の臓器で発現して!/、るマイクロ RNAを同定し、その機能を解析し、疾患と の関係を解明することにより、新しい治療薬及び診断薬が開発されることが期待され 特に、間葉系幹細胞で作用しているマイクロ RNAを見出すことは、間葉系幹細胞に おける分化及び増殖の機能解明につながり、間葉系幹細胞の単離、培養、間葉系 幹細胞から特定の細胞への分化制御法の開発および分化制御を利用した新しい治 療法につながると期待できる。本発明の目的は、間葉系幹細胞で発現しているマイク 口 RNA群を取得し、それらの利用法を提供することにある。 [0006] By identifying microRNAs that are expressed in various human organs !, analyzing their functions, and elucidating their relationship with diseases, new therapeutics and diagnostics may be developed. Expected In particular, the discovery of microRNAs acting on mesenchymal stem cells leads to elucidation of the functions of differentiation and proliferation in mesenchymal stem cells, and isolation, culture, and mesenchymal stem cells from mesenchymal stem cells It can be expected that this will lead to the development of differentiation control methods for specific cells and new therapies using differentiation control. An object of the present invention is to obtain a microphone RNA group expressed in mesenchymal stem cells and provide a method for using them.

課題を解決するための手段  Means for solving the problem

[0007] 本発明は以下の（1 )から（40)に関する。 [0007] The present invention relates to the following (1) to (40).

(1)以下の（a)〜 (j)の!/、ずれかの核酸を有効成分として含有する間葉系幹細胞の 増殖および/または分化抑制剤。  (1) A mesenchymal stem cell proliferation and / or differentiation inhibitor containing the following (a) to (j)! /, Or any nucleic acid as an active ingredient.

(a)配列番号;!〜 93のいずれかで表される塩基配列からなる核酸  (a) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs:! to 93

(b)配列番号 1〜93のいずれかで表される塩基配列を含有する、 15〜28塩基の核 酸 (b) a nucleus of 15 to 28 bases containing the base sequence represented by any of SEQ ID NOs: 1 to 93 acid

(c)配列番号 1〜93のいずれかで表される塩基配列 90%以上の同一性を有する塩 基配列からなる核酸  (c) a nucleic acid comprising a base sequence having 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 93

(d)配列番号 1〜93のいずれかで表される塩基配列からなる核酸の相補鎖とストリン

Figure imgf000005_0001
(d) a complementary strand and string of nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 1 to 93
Figure imgf000005_0001

(e)配列番号;!〜 5のいずれかで表される塩基配列の 2〜8番目の塩基配列を含む 核酸  (e) a nucleic acid comprising the second to eighth base sequences of the base sequence represented by any of SEQ ID NOs:! to 5

(f) (a)〜（e)のいずれかの核酸と、該核酸の塩基配列に対して相補的な塩基配列と からなる核酸とを含む二本鎖核酸  (f) A double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid

(g) (a)〜（e)のいずれかの核酸と、該核酸とストリンジェントな条件でハイブリダィズ する核酸とからなる二本鎖核酸  (g) Double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid that hybridizes with the nucleic acid under stringent conditions

(h)配列番号 94〜201のいずれかで表される塩基配列からなる核酸  (h) a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201

(i)配列番号 94〜201のいずれかで表される塩基配列と 90%以上の同一性を有す る塩基配列からなる核酸  (i) a nucleic acid comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence represented by any of SEQ ID NOS: 94 to 201

(j)配列番号 94〜201のいずれかで表される塩基配列からなる核酸の相補鎖とストリ ンジェントな条件でハイブリダィズする核酸。  (j) A nucleic acid that hybridizes with a complementary strand of a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201 under stringent conditions.

(2) (1)に記載の核酸を発現するベクターを有効成分として含有する間葉系幹細胞 の増殖および/または分化抑制剤。  (2) A mesenchymal stem cell proliferation and / or differentiation inhibitor comprising the vector expressing the nucleic acid according to (1) as an active ingredient.

(3) (1)に記載の核酸の標的遺伝子の発現を抑制する物質を有効成分として含有す る間葉系幹細胞の増殖および/または分化抑制剤。  (3) A mesenchymal stem cell proliferation and / or differentiation inhibitor containing a substance that suppresses the expression of the target gene of the nucleic acid according to (1) as an active ingredient.

(4) ADD3遺伝子または CBFB遺伝子の発現を抑制する物質を有効成分として含 有する間葉系幹細胞の増殖および/または分化抑制剤。  (4) A mesenchymal stem cell proliferation and / or differentiation inhibitor containing a substance that suppresses the expression of ADD3 gene or CBFB gene as an active ingredient.

(5) ADD3遺伝子または CBFB遺伝子の発現を抑制する物質が核酸である（4)に 記載の間葉系幹細胞の増殖および/または分化抑制剤。  (5) The agent for suppressing proliferation and / or differentiation of mesenchymal stem cells according to (4), wherein the substance that suppresses the expression of ADD3 gene or CBFB gene is a nucleic acid.

(6)核酸が siRNAである（5)に記載の間葉系幹細胞の増殖および/または分化抑 制剤。  (6) The agent for suppressing proliferation and / or differentiation of mesenchymal stem cells according to (5), wherein the nucleic acid is siRNA.

(7) siRNAが配列番号 202〜205の!/、ずれかで表される塩基配列を標的配列とす る siRNAである（6)に記載の間葉系幹細胞の増殖および/または分化抑制剤。 (8) (1)に記載の核酸を有効成分として含有する、該核酸の標的遺伝子の発現抑制 剤。 (7) The agent for suppressing proliferation and / or differentiation of mesenchymal stem cells according to (6), wherein siRNA is a siRNA whose target sequence is a nucleotide sequence represented by SEQ ID NOs: 202 to 205! (8) An agent for suppressing expression of a target gene of the nucleic acid, comprising the nucleic acid according to (1) as an active ingredient.

(9)配列番号 3で表される塩基配列からなる核酸または配列番号 97で表される塩基 配列からなる核酸を有効成分とする ADD3遺伝子の発現抑制剤。  (9) An ADD3 gene expression inhibitor comprising as an active ingredient a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 97.

(10)配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基 配列とからなる核酸とを含む二本鎖核酸を有効成分として含有する ADD3遺伝子の 発現抑制剤。  (10) ADD3 gene expression inhibitor comprising as an active ingredient a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid .

(11)配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズ する核酸とからなる二本鎖核酸を有効成分として含有する ADD3遺伝子の発現抑制 剤。  (11) An ADD3 gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.

(12)配列番号 3で表される塩基配列からなる核酸または配列番号 97で表される塩 基配列からなる核酸を有効成分とする CBFB遺伝子の発現抑制剤。  (12) A CBFB gene expression inhibitor comprising as an active ingredient a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 97.

(13)配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基 配列とからなる核酸とを含む二本鎖核酸を有効成分として含有する CBFB遺伝子の 発現抑制剤。  (13) CBFB gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid .

(14)配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズ する核酸とからなる二本鎖核酸を有効成分として含有する CBFB遺伝子の発現抑制 剤。  (14) A CBFB gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising a nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.

(15) (1)に記載の核酸を用いる、間葉系幹細胞の増殖および/または分化抑制方 法。  (15) A method for inhibiting proliferation and / or differentiation of mesenchymal stem cells using the nucleic acid according to (1).

(16) (1)に記載の核酸を発現するベクターを用いる、間葉系幹細胞の増殖および/ または分化抑制方法。  (16) A method for inhibiting the proliferation and / or differentiation of mesenchymal stem cells using the vector expressing the nucleic acid according to (1).

(17) (1)に記載の核酸の標的遺伝子の発現を抑制する物質を用いる間葉系幹細胞 の増殖および/または分化抑制方法。  (17) A method for inhibiting proliferation and / or differentiation of mesenchymal stem cells using a substance that suppresses the expression of a target gene of the nucleic acid according to (1).

(18) ADD3遺伝子または CBFB遺伝子の発現を抑制する物質を用いる間葉系幹 細胞の増殖および/または分化抑制方法。  (18) A method for suppressing the proliferation and / or differentiation of mesenchymal stem cells using a substance that suppresses the expression of ADD3 gene or CBFB gene.

( 19) ADD3遺伝子または CBFB遺伝子の発現を抑制する物質が核酸である（18記 載の間葉系幹細胞の増殖および/または分化抑制方法。 (20)核酸が siRNAである（19)に記載の間葉系幹細胞の増殖および/または分化 抑制方法。 (19) The substance that suppresses the expression of the ADD3 gene or the CBFB gene is a nucleic acid (the method for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to 18). (20) The method for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to (19), wherein the nucleic acid is siRNA.

(21) siRNAが配列番号 202〜205のいずれかで表される塩基配列を標的配列とす る siRNAである（20)に記載の間葉系幹細胞の増殖および/または分化抑制方法。  (21) The method for inhibiting the proliferation and / or differentiation of mesenchymal stem cells according to (20), wherein the siRNA is a siRNA whose target sequence is the base sequence represented by any of SEQ ID NOs: 202 to 205.

(22) (1)に記載の核酸を用いる、該核酸の標的遺伝子の発現抑制方法。  (22) A method for suppressing expression of a target gene of the nucleic acid, using the nucleic acid according to (1).

(23)配列番号 3で表される塩基配列からなる核酸、または配列番号 97で表される塩 基配列のからなる核酸を用いる ADD3遺伝子の発現抑制方法。  (23) A method for suppressing the expression of the ADD3 gene, which uses a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the base sequence represented by SEQ ID NO: 97.

(24)配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基 配列とからなる核酸とを含む二本鎖核酸を用いる ADD3遺伝子の発現抑制方法。  (24) A method for suppressing ADD3 gene expression, comprising using a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid.

(25)配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズ する核酸とからなる二本鎖核酸を用いる ADD3遺伝子の発現抑制方法。  (25) A method for suppressing ADD3 gene expression, comprising using a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.

(26)配列番号 3で表される塩基配列からなる核酸、または配列番号 97で表される塩 基配列からなる核酸を用いる CBFB遺伝子の発現抑制方法。  (26) A method for suppressing the expression of a CBFB gene, which uses a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the base sequence represented by SEQ ID NO: 97.

(27)配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基 配列とからなる核酸とを含む二本鎖核酸を用いる CBFB遺伝子の発現抑制方法。  (27) A method for inhibiting CBFB gene expression using a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid.

(28)配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズ する核酸とからなる二本鎖核酸を用いる CBFB遺伝子の発現抑制方法。  (28) A method for inhibiting CBFB gene expression, comprising using a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions.

(29) (1)に記載の核酸の発現を促進させることを指標とする間葉系幹細胞の増殖お よび/または分化抑制剤のスクリーニング方法。  (29) A method for screening for an agent for suppressing the proliferation and / or differentiation of mesenchymal stem cells using the expression of the nucleic acid according to (1) as an index.

(30) (1)に記載の核酸の標的遺伝子の発現を抑制させることを指標とする間葉系幹 細胞の増殖および/または分化抑制剤のスクリーニング方法。  (30) A screening method for an agent for inhibiting the proliferation and / or differentiation of mesenchymal stem cells, using as an index the suppression of the expression of the target gene of the nucleic acid according to (1).

(31) (1)に記載の核酸の発現を抑制させることを指標とする間葉系幹細胞の増殖お よび/または分化促進剤のスクリーニング方法。  (31) A method for screening for an agent for promoting the proliferation and / or differentiation of mesenchymal stem cells using the expression of the nucleic acid according to (1) as an index.

(32) (1)に記載の核酸の標的遺伝子の発現を促進させることを指標とする間葉系幹 細胞の増殖および/または分化促進剤のスクリーニング方法。  (32) A method for screening an agent for promoting proliferation and / or differentiation of mesenchymal stem cells using the expression of the expression of the target gene of the nucleic acid according to (1) as an index.

(33) (1)に記載の核酸の発現を抑制させる物質を有効成分として含有する間葉系 幹細胞の増殖および/または分化促進剤。  (33) A mesenchymal stem cell proliferation and / or differentiation promoter containing a substance that suppresses the expression of the nucleic acid according to (1) as an active ingredient.

(34)配列番号 1〜201からなる塩基配列と相補的な塩基配列を含む核酸を有効成 分として含有する間葉系幹細胞の増殖および/または分化促進剤。 (34) Effectively producing a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201 A mesenchymal stem cell proliferation and / or differentiation promoter contained as a fraction.

(35) (1)に記載の核酸の発現を抑制させる物質を用いる間葉系幹細胞の増殖およ び/または分化促進方法。  (35) A method for promoting the proliferation and / or differentiation of mesenchymal stem cells using the substance that suppresses the expression of a nucleic acid according to (1).

(36)配列番号 1〜201からなる塩基配列と相補的な塩基配列を含む核酸を用いる 間葉系幹細胞の増殖および/または分化促進方法。  (36) A method for promoting proliferation and / or differentiation of mesenchymal stem cells using a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201.

(37) (1)に記載の核酸、または配列番号 1〜201からなる塩基配列と相補的な塩基 配列を含む核酸を有効成分として含有する、間葉系幹細胞の増殖および/または 分化の異常に起因する疾患の診断薬。  (37) An abnormality in proliferation and / or differentiation of mesenchymal stem cells, containing as an active ingredient the nucleic acid described in (1) or a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence consisting of SEQ ID NOs: 1 to 201 A diagnostic agent for the disease caused.

(38) (1)に記載の核酸の発現、該核酸の変異、該核酸をコードするゲノムの変異を 検出する試薬を有効成分として含有する、間葉系幹細胞の増殖および/または分化 の異常に起因する疾患の診断薬。  (38) Abnormality in proliferation and / or differentiation of mesenchymal stem cells, containing as an active ingredient a reagent for detecting the expression of the nucleic acid, the mutation of the nucleic acid, and the mutation of the genome encoding the nucleic acid described in (1) A diagnostic agent for the disease caused.

(39) (1)に記載の核酸、または配列番号 1〜201からなる塩基配列と相補的な塩基 配列を含む核酸を有効成分として含有する、間葉系幹細胞の増殖および/または 分化の異常に起因する疾患の治療薬。  (39) An abnormality in proliferation and / or differentiation of mesenchymal stem cells, containing as an active ingredient the nucleic acid described in (1) or a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201 A treatment for the disease caused.

(40) (1)〜（7)のいずれかに記載の間葉系幹細胞の増殖および/または分化抑制 剤または（33)または（34)に記載の間葉系幹細胞の増殖および/または分化促進 剤を有効成分として含有する間葉系幹細胞の増殖および/または分化の異常に起 因する疾患の治療薬。  (40) The mesenchymal stem cell proliferation and / or differentiation inhibitor according to any one of (1) to (7) or the mesenchymal stem cell proliferation and / or differentiation promotion according to (33) or (34) A therapeutic agent for diseases caused by abnormal proliferation and / or differentiation of mesenchymal stem cells, which contains the agent as an active ingredient.

発明の効果  The invention's effect

[0008] 本発明により、間葉系幹細胞の増殖および/または分化抑制剤、マイクロ RNAの 標的遺伝子の発現抑制剤、間葉系幹細胞の増殖および/または分化抑制方法、マ イク口 RNAの標的遺伝子の発現抑制方法、間葉系幹細胞の増殖および/または分 化抑制剤のスクリーニング方法、間葉系幹細胞の増殖および/または分化促進剤、 間葉系幹細胞の増殖および/または分化促進方法、間葉系幹細胞の増殖および/ または分化の異常に起因する疾患の診断薬、間葉系幹細胞の増殖および/または 分化の異常に起因する疾患の治療薬を提供することができる。  [0008] According to the present invention, a mesenchymal stem cell proliferation and / or differentiation inhibitor, a microRNA target gene expression inhibitor, a mesenchymal stem cell proliferation and / or differentiation suppression method, and a microphone mouth RNA target gene Expression suppression method, mesenchymal stem cell proliferation and / or differentiation inhibitor screening method, mesenchymal stem cell proliferation and / or differentiation promoting agent, mesenchymal stem cell proliferation and / or differentiation promoting method, mesenchyme It is possible to provide a diagnostic agent for diseases caused by abnormal stem cell proliferation and / or differentiation and a therapeutic agent for diseases caused by abnormal mesenchymal stem cell proliferation and / or differentiation.

図面の簡単な説明  Brief Description of Drawings

[0009] [図 1]図 1は、 hMSCの骨芽細胞分化過程におけるマイクロ RNAの発現量変動を示し た図であり、 Aは hsa_miR-100、 Bは hsa_miR-145、 Cは hsa_miR-210、 Dは hsa-miR-4 22bの発現変動を示した図である。横軸は、培養日数を示し、縦軸は、分化誘導前の hMSCにおいて発現している各マイクロ RNAの発現量を 1.0として、相対発現量を表す[0009] Fig. 1 shows changes in microRNA expression during hMSC osteoblast differentiation. A shows hsa_miR-100, B shows hsa_miR-145, C shows hsa_miR-210, and D shows hsa-miR-4 22b. The horizontal axis represents the number of culture days, and the vertical axis represents the relative expression level, where 1.0 is the expression level of each microRNA expressed in hMSC before differentiation induction.

〇 Yes

発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION

[0010] 本発明の間葉系幹細胞の増殖および/または分化抑制剤に用いられる核酸として は、以下の核酸があげられる。該核酸は、マイクロ RNAまたはその誘導体、マイクロ RNA前駆体またはその誘導体および二本鎖核酸 (以下、本発明に用いられる核酸 ともレ、う）であることが好ましレ、。 [0010] Examples of the nucleic acid used for the agent for suppressing proliferation and / or differentiation of mesenchymal stem cells of the present invention include the following nucleic acids. The nucleic acid is preferably microRNA or a derivative thereof, a microRNA precursor or a derivative thereof and a double-stranded nucleic acid (hereinafter also referred to as a nucleic acid used in the present invention).

(a)配列番号;!〜 93のいずれかで表される塩基配列からなる核酸  (a) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs:! to 93

(b)配列番号 1〜93のいずれかで表される塩基配列を含有する、 15〜28塩基の核 酸  (b) a 15-28 base nucleic acid containing the base sequence represented by any of SEQ ID NOs: 1-93

(c)配列番号 1〜93のいずれかで表される塩基配列 90%以上の同一性を有する塩 基配列からなる核酸  (c) a nucleic acid comprising a base sequence having 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 93

(d)配列番号 1〜93のいずれかで表される塩基配列からなる核酸の相補鎖とストリン ジェントな条件でハイブリダィズする核酸  (d) a nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid comprising the nucleotide sequence represented by any of SEQ ID NOs: 1 to 93

(e)配列番号;!〜 5のいずれかで表される塩基配列の 2〜8番目の塩基配列を含む 核酸  (e) a nucleic acid comprising the second to eighth base sequences of the base sequence represented by any of SEQ ID NOs:! to 5

(f) (a)〜（e)のいずれかの核酸と、該核酸の塩基配列に対して相補的な塩基配列と からなる核酸とを含む二本鎖核酸  (f) A double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid

(g) (a)〜（e)のいずれかの核酸と、該核酸とストリンジェントな条件でハイブリダィズ する核酸とからなる二本鎖核酸  (g) Double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid that hybridizes with the nucleic acid under stringent conditions

(h)配列番号 94〜201のいずれかで表される塩基配列からなる核酸  (h) a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201

(i)配列番号 94〜201のいずれかで表される塩基配列と 90%以上の同一性を有す る塩基配列からなる核酸  (i) a nucleic acid comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence represented by any of SEQ ID NOS: 94 to 201

(j)配列番号 94〜201のいずれかで表される塩基配列からなる核酸の相補鎖とストリ ンジェントな条件でハイブリダィズする核酸。  (j) A nucleic acid that hybridizes with a complementary strand of a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201 under stringent conditions.

[0011] 本発明においてマイクロ RNAとは、細胞内に存在する一本鎖 RNAであり、且つ、該 配列を含む周辺ゲノム配列がヘアピン構造を形成し得る配列を有しており、ヘアピン のいずれか片鎖カも切り出され得る RNAをいう。マイクロ RNAの長さとしては、 15〜2 8塩基が好ましぐ 16〜28塩基がより好ましぐ 16〜26塩基がさらに好ましぐ 16〜2 4塩基が特に好ましい。マイクロ RNAは、その標的となる mRNAに相補的に結合して m RNAの分解あるいは翻訳を抑制し、遺伝子発現の転写後制御を行う。本発明で用い るマイクロ RNAとして、例えば、配列番号;!〜 5のいずれかで表される塩基配列から なるヒトマイクロ RNAをあげることができる。さらに、配列番号 1〜5のいずれかで表さ れる塩基配列からなるヒトマイクロ RNAと同一の機能を有するマイクロ RNAとして、 該ヒトマイクロ RNAのォーソログである、配列番号 6〜65で表される塩基配列からな る核酸をあげること力できる。具体的な例として、配列番号 1のヒトマイクロ RNAのォ ーソログは配列番号 6〜20、配列番号 2のヒトマイクロ RNAのォーソログは配列番号 21—40,配列番号 3のヒトマイクロ RNAのォーソログは配列番号 4；!〜 52、配列番 号 4のヒトマイクロ RNAのォーソログは配列番号 53〜63、配列番号 5のヒトマイクロ R NAのォーソログは配列番号 64〜65でそれぞれ表される塩基配列からなる核酸をあ げること力 Sできる。また、マイクロ RNAがその標的遺伝子の mRNAの翻訳を抑制する メカニズムとして、マイクロ RNAの 5'末端側 2〜8番目の塩基配列に対して相補的な 塩基配列を有する mRNAは、マイクロ RNA標的遺伝子として認識され、その翻訳が マイクロ RNAにより抑制されることが知られている [Current Biology, 15， R458-R460 ( 2005)]。従って、配列番号 1〜5で表されるヒトマイクロ RNAの 5'末端側 2〜8番目力 S 同じ塩基配列を有するマイクロ RNAは、同じ mRNAの翻訳を抑制して同様の機能を 有する。該マイクロ RNAの具体的な例としては、配列番号 1のマイクロ RNAに対して は、配列番号 66〜67、配列番号 2のマイクロ RNAに対しては配列番号 68〜92、配 列番号 5のマイクロ RNAに対しては配列番号 93でそれぞれ表される塩基配列からな る核酸をあげること力でさる。 [0011] In the present invention, the microRNA is a single-stranded RNA present in a cell, and the The surrounding genomic sequence including the sequence has RNA that can form a hairpin structure, and it means RNA that can cut out any one strand of the hairpin. The length of the microRNA is preferably 15 to 28 bases, more preferably 16 to 28 bases, more preferably 16 to 26 bases, and particularly preferably 16 to 24 bases. MicroRNA binds complementarily to its target mRNA, suppresses mRNA degradation or translation, and controls post-transcriptional gene expression. Examples of the microRNA used in the present invention include human microRNA having a base sequence represented by any of SEQ ID NOs:! -5. Furthermore, as a microRNA having the same function as the human microRNA consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 5, the base represented by SEQ ID NOs: 6 to 65, which is an ortholog of the human microRNA It is possible to raise nucleic acids consisting of sequences. As a specific example, the human microRNA ortholog of SEQ ID NO: 1 is SEQ ID NO: 6-20, the human microRNA ortholog of SEQ ID NO: 2 is SEQ ID NO: 21-40, and the human microRNA ortholog of SEQ ID NO: 3 is a sequence No. 4;! -52, the human microRNA ortholog of SEQ ID NO: 4 is a nucleic acid comprising the nucleotide sequences represented by SEQ ID NOs: 53-63, and the human microRNA of SEQ ID NO: 5 is represented by SEQ ID NOs: 64-65, respectively. The ability to raise S In addition, as a mechanism by which microRNA suppresses translation of mRNA of its target gene, mRNA having a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is used as a microRNA target gene. It is known that its translation is suppressed by microRNA [Current Biology, 15, R458-R460 (2005)]. Accordingly, the second to eighth forces on the 5 ′ end side of the human microRNA represented by SEQ ID NOs: 1 to 5 S The microRNA having the same base sequence has the same function by suppressing translation of the same mRNA. Specific examples of the microRNA include SEQ ID NO: 66 to 67 for the microRNA of SEQ ID NO: 1, and SEQ ID NO: 68 to 92 for the microRNA of SEQ ID NO: 2, and the microRNA of SEQ ID NO: 5. For RNA, it refers to the ability to raise a nucleic acid consisting of the base sequence represented by SEQ ID NO: 93.

本発明において、マイクロ RNA前駆体とは、本発明に用いるマイクロ RNAを含む約 50〜約 200塩基長、より好ましくは約 70〜約 100塩基長の核酸であり、且つ、ヘア ピン構造を形成し得る核酸である。 Dicerと呼ばれる蛋白質によるプロセシングを経て 、マイクロ RNA前駆体からマイクロ RNAが生成される。本発明で用いるマイクロ RN A前駆体として、例えば、配列番号 1のヒトマイクロ RNAに対しては配列番号 94〜95 、配列番号 2のヒトマイクロ RNAに対しては配列番号 96、配列番号 3のヒトマイクロ R NAに対しては配列番号 97、配列番号 4のヒトマイクロ RNAに対しては配列番号 98 、配列番号 5のヒトマイクロ RNAに対しては配列番号 99でそれぞれ表される塩基配 歹 IJ力、らなる核酸をあげること力 Sできる。さらに、ォーソログのマイクロ RNAの前駆体もヒ トマイクロ RNAの前駆体と同様に機能するので、本発明で用いるマイクロ RNA前駆 体としてあげること力 Sできる。具体的な例として、配列番号 6〜20のいずれかで表され る配列番号 1のヒトマイクロ RNAのォーソログに対するマイクロ RNA前駆体として配 歹 IJ番号 100〜； 126、配列番号 2；!〜 40のいずれかで表される配列番号 2のヒトマイク 口 RNAのォーソログに対するマイクロ RNA前駆体として配列番号 127〜147、配列 番号 4；!〜 52のいずれかで表される配列番号 3のヒトマイクロ RNAのォーソログに対 するマイクロ RNA前駆体として配列番号 148〜159、配列番号 53〜63のいずれか で表される配列番号 4のヒトマイクロ RNAのォーソログに対するマイクロ RNA前駆体 として酉己歹 IJ番号 160〜； 170、配列番号 64〜65のいずれかで表される配列番号 5のヒ トマイクロ RNAのォーソログに対するマイクロ RNA前駆体として配列番号 17；!〜 172 でそれぞれ表される塩基配列からなる核酸をあげることができる。また、配列番号；!〜 5で表されるヒトマイクロ RNAの 5'末端側 2〜8番目が同じ塩基配列を有するマイクロ RNAは、同じ mRNAの翻訳を抑制して同様の機能を有すると考えられるので、該マイ クロ RNAの前駆体も本発明で用いるマイクロ RNA前駆体としてあげることができる。 具体的な例として、配列番号 66〜67のいずれかで表されるマイクロ RNAに対しては 、配列番号 173〜； 174のいずれかで表される塩基配列からなる核酸、配列番号 68 〜92のいずれかで表されるマイクロ RNAに対しては配列番号 175〜200のいずれ かで表される塩基配列からなる核酸、配列番号 93で表されるマイクロ RNAに対して は配列番号 201で表される塩基配列からなる核酸をあげることができる。 In the present invention, the microRNA precursor is a nucleic acid having a length of about 50 to about 200 bases, more preferably about 70 to about 100 bases including the microRNA used in the present invention, and forms a hairpin structure. The nucleic acid to be obtained. MicroRNA is produced from the microRNA precursor through processing by a protein called Dicer. Micro RN used in the present invention As the A precursor, for example, SEQ ID NO: 94 to 95 for the human microRNA of SEQ ID NO: 1, SEQ ID NO: 96 for the human microRNA of SEQ ID NO: 2, against the human microRNA of SEQ ID NO: 3 Is the nucleotide sequence IJ force represented by SEQ ID NO: 98 for the human microRNA of SEQ ID NO: 97 and SEQ ID NO: 4, and SEQ ID NO: 99 for the human microRNA of SEQ ID NO: 5, respectively. That power S. Furthermore, the ortholog microRNA precursor functions in the same manner as the human microRNA precursor, and can therefore be used as a microRNA precursor for use in the present invention. As a specific example, as a microRNA precursor to an ortholog of the human microRNA of SEQ ID NO: 1 represented by any one of SEQ ID NOS: 6-20, IJ numbers 100-; 126, SEQ ID NO: 2; A human microRNA ortholog of SEQ ID NO: 3 represented by any one of SEQ ID NO: 127 to 147, SEQ ID NO: 4;! -52 as a microRNA precursor to a human microphone RNA ortholog of SEQ ID NO: 2 represented by As a microRNA precursor for human microRNA ortholog of SEQ ID NO: 4 represented by any one of SEQ ID NO: 148-159 and SEQ ID NO: 53-63 as a microRNA precursor for IJ No. 160-170; , As a microRNA precursor to the human microRNA ortholog of SEQ ID NO: 5 represented by any of SEQ ID NOs: 64 to 65, comprising a nucleotide sequence represented by SEQ ID NO: 17;! To 172, respectively. It is possible to increase the nucleic acid. In addition, it is considered that microRNAs having the same nucleotide sequence at the 5 'end side of the human microRNA represented by SEQ ID NOs:! To 5 have the same function by suppressing the translation of the same mRNA. Therefore, the precursor of the microRNA can also be mentioned as the microRNA precursor used in the present invention. As a specific example, for the microRNA represented by any of SEQ ID NOs: 66 to 67, a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 173 to 174, SEQ ID NOs: 68 to 92 For the microRNA represented by any one, a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 175 to 200, and for the microRNA represented by SEQ ID NO: 93, represented by SEQ ID NO: 201 A nucleic acid consisting of a base sequence can be mentioned.

本発明において、配列番号;!〜 201のいずれかで表される塩基配列と 90%以上の 同一性を有する核酸とは、 BLAST [J. Mol. Biol., 215， 403 (1990)〕や FASTA〔Met hods in Enzymology, 183， 63 (1990)〕等の解析ソフトを用いて計算したときに、配列番 号 1〜201のいずれかで表される塩基配列からなる核酸と少なくとも 90%以上、好ま しくは 93%以上、より好ましくは 95%以上、さらに好ましくは 96%以上、特に好ましく は 97%以上、最も好ましくは 98%以上である核酸であることを意味する。 In the present invention, a nucleic acid having 90% or more identity with the base sequence represented by any of SEQ ID NOs:! To 201 is BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA. When calculated using analysis software such as [Met hods in Enzymology, 183, 63 (1990)], at least 90% or more of the nucleic acid consisting of the nucleotide sequence represented by any of SEQ ID NOs: 1-201 is preferred. Or 93% or more, more preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more.

[0014] またストリンジェントな条件とは、一方の鎖をブロットしたメンブレンに対し、 7.5 mL、 1 M Na HPO (pH7.2) 0.6 mL、 10% SDS 21 mL、 50xDenhardt's solution 0.6 mL、 10 mg [0014] In addition, stringent conditions are 7.5 mL, 1 M Na HPO (pH 7.2) 0.6 mL, 10% SDS 21 mL, 50x Denhardt's solution 0.6 mL, 10 mg on the membrane blotted on one strand.

2 4  twenty four

/mL sonicated salmon sperm DNA 0.3 mL力、ら成る Hybridization bufferに、 P—ATP で標識した他方の鎖を加え、 50°Cでー晚反応させた後、 50°Cで 10分間、 5xSSC/5%S DS液で洗浄し、更に 50°Cで 10分間、 lxSSC/l%SDS液で洗浄し、その後メンブレンを 取り出し、 X線フィルムに感光させることによりシグナルを検出できる条件である。  Add the other strand labeled with P-ATP to a hybridization buffer consisting of 0.3 mL of sonicated salmon sperm DNA, and react at 50 ° C for 10 minutes at 5 ° SSC / 5%. Washing with SDS solution, and further washing with lxSSC / l% SDS solution at 50 ° C for 10 minutes, then taking out the membrane and exposing it to X-ray film are the conditions under which signals can be detected.

[0015] 本発明において、核酸とはヌクレオチドおよび該ヌクレオチドと同等の機能を有する 分子が重合した分子であればいかなるものでもよぐ例えば、リボヌクレオチドの重合 体である RNA、デォキシリボヌクレオチドの重合体である DNA、 RNAおよび DNAが混 合した重合体、および、ヌクレオチド類似体を含むヌクレオチド重合体をあげることが でき、さらに、核酸誘導体を含むヌクレオチド重合体であってもよぐ一本鎖核酸また は二本鎖核酸であってもよい。またマイクロ RNAまたはその誘導体、マイクロ RNA前 駆体またはその誘導体も本発明の核酸に含まれる。  In the present invention, the nucleic acid may be any molecule in which nucleotides and molecules having functions equivalent to the nucleotides are polymerized. For example, RNA, which is a polymer of ribonucleotides, and deoxyribonucleotides. Examples of the polymer include DNA, RNA and a mixture of RNA and nucleotide, and nucleotide polymers including nucleotide analogs, and may be nucleotide polymers including nucleic acid derivatives. It may be a nucleic acid or a double-stranded nucleic acid. MicroRNA or a derivative thereof, a microRNA precursor or a derivative thereof are also included in the nucleic acid of the present invention.

[0016] 本発明にお!/、て、ヌクレオチド類似体とは、 RNAまたは DNAに比べ、ヌクレアーゼ耐 性の向上または、安定化させるため、相補鎖核酸とのァフィ二ティーをあげるため、あ るいは細胞透過性をあげるため、あるいは可視化させるために、リボヌクレオチド、デ ォキシリボヌクレオチド、 RNAまたは DNAに修飾を施した分子であれば!/、かなる分子 でもよく、天然に存在する分子でも非天然の分子でもよぐ例えば、糖部修飾ヌクレオ チド類似体やリン酸ジエステル結合修飾ヌクレオチド類似体等があげられる。  [0016] In the present invention, a nucleotide analog is used to increase the affinity with a complementary strand nucleic acid in order to improve or stabilize nuclease resistance compared to RNA or DNA. Can be any molecule that modifies ribonucleotides, doxyribonucleotides, RNA or DNA in order to increase cell permeability or to make it visible! Examples include non-natural molecules such as sugar-modified nucleotide analogs and phosphodiester bond-modified nucleotide analogs.

[0017] 糖部修飾ヌクレオチド類似体とは、ヌクレオチドの糖の化学構造の一部あるいは全 てに対し、任意の化学構造物質を付加あるいは置換したものであればいかなるもの でもよく、例えば、 2' 0—メチルリボースで置換されたヌクレオチド類似体、 2'— 0 プロピルリボースで置換されたヌクレオチド類似体、 2，ーメトキシエトキシリボースで 置換されたヌクレオチド類似体、 2'— 0—メトキシェチルリボースで置換されたヌクレ ォチド類似体、 2'— 0— [2—（グァニジゥム）ェチル]リボースで置換されたヌクレオチ ド類似体、 2' 0—フルォロリボースで置換されたヌクレオチド類似体、糖部に架橋 構造を導入することにより 2つの環状構造を有する架橋構造型人工核酸 (Bridged Nu oleic Acid) (BNA)、より具体的には、 2 '位の酸素原子と 4 '位の炭素原子がメチレン を介して架橋したロックト人工核酸 (Locked Nucleic Acid) (LNA)、およびエチレン 架橋構造型人工核酸（Ethylene bridged nucleic acid) (ENA) [Nucleic Acid Researc h， 32， el75(2004)]があげられ、さらにペプチド核酸（PNA) [Acc. Chem. Res., 32， 62 4 (1999)]、ォキシペプチド核酸（O PNA) [J. Am. Chem. Soc , 123， 4653 (2001)]、お よびペプチドリボ核酸（PRNA) [J. Am. Chem. Soc, 122， 6900 (2000)]等をあげること ができる。 [0017] The sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting an arbitrary chemical structural substance to a part or all of the chemical structure of the sugar of the nucleotide. Nucleotide analogs substituted with 0-methylribose, nucleotide analogs substituted with 2'-0 propyl ribose, nucleotide analogs substituted with 2, -methoxyethoxyribose, 2'- 0-methoxyethyl ribose Substituted nucleotide analogs, nucleotide analogs substituted with 2'-0- [2- (guanidinium) ethyl] ribose, nucleotide analogs substituted with 2 '0-fluororibose, cross-linked to the sugar moiety Bridged Nucleic Acid (BNA) having two circular structures by introducing a structure, more specifically, a 2′-position oxygen atom and a 4′-position carbon atom via methylene. Locked Nucleic Acid (LNA) and Ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, el75 (2004)], and peptides Nucleic acid (PNA) [Acc. Chem. Res., 32, 62 4 (1999)], Oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc, 123, 4653 (2001)], and peptide ribonucleic acid ( PRNA) [J. Am. Chem. Soc, 122, 6900 (2000)].

[0018] リン酸ジエステル結合修飾ヌクレオチド類似体とは、ヌクレオチドのリン酸ジエステ ル結合の化学構造の一部あるいは全てに対し、任意の化学物質を付加ある!/ヽは置 換したものであればいかなるものでもよぐ例えば、ホスフォロチォエート結合に置換 されたヌクレオチド類似体、 Ν3'_Ρ5'ホスフォアミデート結合に置換されたヌクレオチド 類似体等をあげること力できる。別の核酸の誘導体として、例えば、糖の修飾体として は、 2'-0_プロピルリボースで置換されたオリゴヌクレオチド誘導体、 2'—メトキシエト キシリボースで置換されたオリゴヌクレオチド誘導体、 2， -0-メチルリボースで置換さ れたオリゴヌクレオチド誘導体、 2， -0-メトキシェチルリボースで置換されたオリゴヌク レオチド誘導体、 2，-0- [2- (グァニジゥム）ェチル]リボースで置換されたオリゴヌタレ ォチド誘導体、 2 ' -0-フルォロリボースで置換されたオリゴヌクレオチド誘導体等をあ げること力 Sでき、リン酸基の修飾体としては、オリゴヌクレオチド中のリン酸ジエステル 結合がホスフォロチォエート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌク レオチド中のリン酸ジエステル結合が Ν3'_Ρ5'ホスフォアミデート結合に変換されたォ リゴヌクレオチド誘導体等をあげることができる [細胞工学， 16， 1463-1473 (1997)] [R NAi法とアンチセンス法、講談社 (2005)]。  [0018] Phosphodiester bond-modified nucleotide analogs are those in which any chemical substance is added to part or all of the chemical structure of a phosphodiester bond of a nucleotide! For example, nucleotide analogues substituted with phosphorothioate bonds, nucleotide analogues substituted with Ν3'_Ρ5 'phosphoramidate linkages, and the like can be mentioned. As other nucleic acid derivatives, for example, modified sugars include oligonucleotide derivatives substituted with 2'-0_propyl ribose, oligonucleotide derivatives substituted with 2'-methoxyethoxyribose, 2, -0-methyl Oligonucleotide derivatives substituted with ribose, oligonucleotide derivatives substituted with 2, -0-methoxyethyl ribose, oligonucleotide derivatives substituted with 2, -0- [2- (guanidinium) ethyl] ribose, 2 'It is possible to raise oligonucleotide derivatives substituted with -0-fluororibose, and the modified phosphate group is an oligonucleotide in which the phosphate diester bond in the oligonucleotide is converted to a phosphoroate bond. Nucleotide derivatives and phosphodiester bonds in oligonucleotides are Ν3'_Ρ5 'phosphoramidate bonds And the like. [Cell engineering, 16, 1463-1473 (1997)] [RNAi method and antisense method, Kodansha (2005)].

[0019] 本発明において、核酸誘導体とは、核酸に比べ、ヌクレアーゼ耐性を向上させるた め、安定化させるため、相補鎖核酸とのァフィ二ティーをあげるため、細胞透過性を あげるため、あるいは可視化させるために、該核酸に別の化学物質を付加した分子 であればいかなる分子でもよぐ例えば、 5 ' ポリアミン付加誘導体、コレステロール 付加誘導体、ステロイド付加誘導体、胆汁酸付加誘導体、ビタミン付加誘導体、 Cy5 付加誘導体、 Cy3付加誘導体、 6— FAM付加誘導体、およびビォチン付加誘導体等 をあげることができる。 [0019] In the present invention, the nucleic acid derivative is used for improving nuclease resistance, stabilizing, increasing affinity with a complementary strand nucleic acid, increasing cell permeability, or visualization compared to nucleic acid. For example, 5 'polyamine addition derivative, cholesterol addition derivative, steroid addition derivative, bile acid addition derivative, vitamin addition derivative, Cy5 Addition derivatives, Cy3 addition derivatives, 6-FAM addition derivatives, biotin addition derivatives, and the like.

[0020] 本発明において、マイクロ RNA誘導体とは、該マイクロ RNAと同等の機能を有す る分子が重合した分子であればいかなるものでもよぐ例えば、リボヌクレオチドの重 合体である RNA、デォキシリボヌクレオチドの重合体である DNA、 RNAおよび DNAが 混合した重合体、および、ヌクレオチド類似体を含むヌクレオチド重合体をあげること ができ、さらに、核酸誘導体を含むヌクレオチド重合体であってもよぐ一本鎖核酸ま たは二本鎖核酸であってもよ!/、。  [0020] In the present invention, the microRNA derivative may be any molecule in which a molecule having a function equivalent to that of the microRNA is a polymerized molecule. Examples of the DNA polymer include DNA, RNA and DNA mixed polymers, and nucleotide polymers including nucleotide analogs. In addition, nucleotide polymers including nucleic acid derivatives may be used. Can be single-stranded or double-stranded nucleic acid! /.

[0021] マイクロ RNA前駆体の誘導体とは、該マイクロ RNA前駆体と同等の機能を有する 分子が重合した分子であればいかなるものでもよぐ例えば、リボヌクレオチドの重合 体である RNA、デォキシリボヌクレオチドの重合体である DNA、 RNAおよび DNAが混 合した重合体、および、ヌクレオチド類似体を含むヌクレオチド重合体をあげることが でき、さらに、核酸誘導体を含むヌクレオチド重合体であってもよぐ一本鎖核酸また は二本鎖核酸であってもよレ、。  [0021] The derivative of the microRNA precursor may be any molecule in which a molecule having a function equivalent to that of the microRNA precursor is polymerized. For example, RNA or deoxy that is a polymer of ribonucleotides Examples of the ribonucleotide polymer include DNA, a polymer in which RNA and DNA are mixed, and a nucleotide polymer including a nucleotide analog, and may be a nucleotide polymer including a nucleic acid derivative. It can be a single-stranded nucleic acid or a double-stranded nucleic acid.

[0022] 間葉系幹細胞とは、骨髄、脂肪組織、臍帯血、子宮内膜、真皮、骨格筋、骨膜、歯 小嚢、歯根膜、歯髄、および歯胚等の間葉系組織に存在し、少なくとも骨芽細胞、脂 肪細胞、および筋肉細胞等の間葉系細胞への分化能を有する細胞をいう。  [0022] Mesenchymal stem cells are present in mesenchymal tissues such as bone marrow, adipose tissue, umbilical cord blood, endometrium, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, pulp, and tooth germ. In other words, it refers to a cell having the ability to differentiate into mesenchymal cells such as at least osteoblasts, fat cells, and muscle cells.

本発明に用いられるマイクロ RNAまたはその誘導体、マイクロ RNA前駆体または その誘導体、または二本鎖核酸を製造する方法としては、特に限定されず、公知の 化学合成を用いる方法、あるいは、酵素的転写法等にて製造することができる。公知 の化学合成を用いる方法として、ホスホロアミダイト法、ホスフォロチォエート法、ホス ホトリエステル法等をあげることができ、例えば、 ABI3900ハイスループット核酸合成 機（アプライドバイォシステムズ社製）により合成すること力 Sできる。酵素的転写法とし ては、 目的の塩基配列を有したプラスミドまたは DNAを铸型として典型的なファージ R NAポリメラーゼ、例えば、 T7、 Τ3、または SP6RNAポリメラーゼを用いた転写法をあげ ること力 Sでさる。  The method for producing the microRNA or derivative thereof, microRNA precursor or derivative thereof, or double-stranded nucleic acid used in the present invention is not particularly limited, and is a method using a known chemical synthesis or an enzymatic transcription method. Etc. can be manufactured. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, and phosphorotriester method. For example, the synthesis is performed using an ABI3900 high-throughput nucleic acid synthesizer (Applied by Systems). That power S. As an enzymatic transcription method, a transcription method using a typical phage RNA polymerase, for example, T7, Τ3, or SP6 RNA polymerase, using a plasmid or DNA having the target nucleotide sequence as a cage type S I'll do it.

[0023] 本発明に用いられるマイクロ RNAまたはマイクロ RNA前駆体の発現を検出する方 法としては、検体中のマイクロ RNAあるいはまたはマイクロ RNA前駆体の存在が検出 できる方法であれば、いかなる方法でもよぐ例えば、（1)ノーザンハイブリダィゼーシ ヨン、 （2)ドットブロットハイブリダィゼーシヨン、 （3) in situハイブリダィゼーシヨン、 （4) 定量的 PCR、 （5)デフアレンシャル 'ノヽイブリダィゼーシヨン、（6)マイクロアレイ、（7)リ ボヌクレアーゼ保護アツセィ等が挙げられる。 [0023] As a method for detecting the expression of microRNA or microRNA precursor used in the present invention, the presence of microRNA or microRNA precursor in a sample is detected. For example, (1) Northern hybridization, (2) Dot blot hybridization, (3) In situ hybridization, (4) Quantification PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay, and the like.

[0024] 本発明で用いるマイクロ RNAまたはマイクロ RNA前駆体の変異を検出する方法、 及び、マイクロ RNAまたはマイクロ RNA前駆体をコードするゲノムの変異を検出する 方法としては、検体中のマイクロ RNAあるいはまたはマイクロ RNA前駆体の塩基配 列の変異が検出できる方法であれば、いかなる方法でもよぐ例えば、非変異型塩基 配列を有する核酸と変異型塩基配列を有する核酸とのハイブリダィズにより形成され るへテロ二本鎖を検出する方法や、あるいは、検体由来の塩基配列を直接、配列決 定して変異の有無を検出する方法等をあげることができる。 [0024] As a method for detecting a mutation in microRNA or a microRNA precursor used in the present invention and a method for detecting a mutation in a genome encoding microRNA or a microRNA precursor, microRNA in a sample or or Any method can be used as long as it can detect a mutation in the base sequence of the microRNA precursor. For example, a heterogeneous complex formed by hybridization of a nucleic acid having a non-mutated base sequence and a nucleic acid having a mutant base sequence. Examples thereof include a method for detecting a double strand, and a method for detecting the presence or absence of mutation by directly sequencing a base sequence derived from a specimen.

[0025] 本発明に用いるマイクロ RNAまたはその誘導体、マイクロ RNA前駆体またはその 誘導体、または二本鎖核酸を発現するベクターとは、細胞内に導入して転写されるこ とにより該核酸が生合成されるように設計されたベクターをいい、細胞内で該マイクロ RNAまたはその誘導体、マイクロ RNA前駆体またはその誘導体、または二本鎖核 酸を転写することができるプロモーターを有して!/、ればレ、かなるベクターでもよ!/、。具 体的には、 PCDNA6.2- GW/miR (Invitrogen社製）、 pSilencer 4.1- CMV(Ambion社製 )、 pSINsi-hHl DNA (タカラバイオ社製）、 pSINsi_hU6 DNA (タカラバイオ社製）、 pEN TR/U6 (Invitrogen社製）等をあげることができる。  [0025] The microRNA used in the present invention or a derivative thereof, a microRNA precursor or a derivative thereof, or a vector expressing a double-stranded nucleic acid is introduced into a cell and transcribed to biosynthesize the nucleic acid. A vector designed to have a promoter capable of transcribing the microRNA or a derivative thereof, a microRNA precursor or a derivative thereof, or a double-stranded nucleic acid in a cell! / It ’s a good vector! Specifically, PCDNA6.2-GW / miR (Invitrogen), pSilencer 4.1-CMV (Ambion), pSINsi-hHl DNA (Takara Bio), pSINsi_hU6 DNA (Takara Bio), pEN TR / U6 (manufactured by Invitrogen) can be listed.

[0026] 標的遺伝子とは、本発明で用いるマイクロ RNAによって認識される数塩基からなる 核酸の塩基配列で、且つ、該塩基配列を有する mRNAの翻訳が該マイクロ RNAによ り抑制される塩基配列を有する遺伝子を!/、う。マイクロ RNAの 5'末端側 2〜8番目の 塩基配列に対して相補的な塩基配列は、該塩基配列を有する mRNAが該マイクロ R NAによって翻訳が抑制される [Current Biology, 15, R458-R460 (2005)]ので、本発 明で用いるマイクロ RNAの 5'末端側 2〜8番目の塩基配列に対して相補的な塩基配 列を、該マイクロ RNAにより抑制される塩基配列としてあげることができる。標的遺伝 子は、例えばマイクロ RNAの 5'末端側 2〜8番目の塩基配列に対して相補的な塩基 配列（以下、マイクロ RNAの標的塩基配列ともいう）を用意し、ヒト mRNAの 3'UTR塩 基配列群に対して、完全一致配列を含有する mRNAを、文字列探索などの方法で選 抜することで決定すること力できる。ヒト mRNAの 3'UTR塩基配列群は、「UCSC Huma n Genome Browser Gateway (http:// genome.ucsc.edu/cgi— bin/hgGatewayノ」より取 得できるゲノム配列および遺伝子位置情報を用いて作製することができる。配列番号 ；!〜 5で表されるマイクロ RNAの標的遺伝子の具体的な例としては、米国国立バイオ テクノロジ■ ~'晴報センター National Center for Biotechnology Information(NCBI)の En treGeneデータベース (http://www.ncbi.nlm.nih.gov/Elntrez/)で ^ [吏われて!/、る名冃 ij ( Official Symbolと Gene ID)で表記された表 1に記載の標的遺伝子をあげることができ る。遺伝子名は、 EntreGeneデータベースの 2006年 3月時点での名前を使用した。ま た、本発明で用いられるマイクロ RNAとして含まれる該ヒトマイクロ RNAのォーソログ および該ヒトマイクロ RNAの 5 '末端側 2〜8番目が同じ塩基配列を有するマイクロ RN Aの標的遺伝子としては、該ヒトマイクロ RNAの標的遺伝子と同一となる。具体的に は、配列番号 6〜20、 66〜67のいずれかで表される塩基配列からなる核酸の標的 遺伝子としては、表 1において配列番号 1のマイクロ RNAの標的遺伝子と同一遺伝 子があげられる。また、配列番号 21〜40、 68〜92のいずれかで表される塩基配列 からなる核酸の標的遺伝子としては、表 1において配列番号 2のマイクロ RNAの標的 遺伝子と同一の遺伝子をあげることができる。また、配列番号 4；!〜 52のいずれかで 表される塩基配列からなる核酸の標的遺伝子としては、表 1において配列番号 3のマ イク口 RNAの標的遺伝子と同一の遺伝子をあげることができる。また、配列番号 53 〜63のいずれかで表される塩基配列からなる核酸の標的遺伝子としては、表 1にお いて配列番号 4のマイクロ RNAの標的遺伝子と同一の遺伝子をあげることができる。 また、配列番号 64〜65、 93のいずれかで表される塩基配列からなる核酸の標的遺 伝子としては、表 1において配列番号 5のマイクロ RNAの標的遺伝子と同一の遺伝 子をあげることができる。 [0026] The target gene is a nucleic acid base sequence consisting of several bases recognized by the microRNA used in the present invention, and a base sequence in which translation of mRNA having the base sequence is suppressed by the microRNA. A gene with! The base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is that translation of mRNA having the base sequence is suppressed by the microRNA [Current Biology, 15, R458-R460 (2005)], a base sequence complementary to the 2-8th base sequence on the 5 ′ end side of the microRNA used in the present invention can be listed as a base sequence to be suppressed by the microRNA. . For the target gene, for example, a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA (hereinafter also referred to as the target base sequence of microRNA) is prepared, and the 3'UTR of human mRNA is prepared. salt It is possible to determine by selecting a mRNA containing a completely identical sequence from a base sequence group by a method such as character string search. The 3'UTR base sequence group of human mRNA is created using the genome sequence and gene position information that can be obtained from UCSC Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway) Specific examples of microRNA target genes represented by SEQ ID NOs:! To 5 include US National Biotechnology ■ ~ 'Gare Information Center National Center for Biotechnology Information (NCBI) En treGene database (http://www.ncbi.nlm.nih.gov/Elntrez/) ^ (Recognized! /, the name ij (Official Symbol and Gene ID) The name of the gene used was the name of the EntreGene database as of March 2006. Also, the human microRNA ortholog and the human microRNA included in the present invention as the microRNA are used. 5 'microphone whose 2nd to 8th ends have the same nucleotide sequence (B) The target gene of RNA is the same as the target gene of the human microRNA, specifically, the target gene of a nucleic acid comprising the base sequence represented by any one of SEQ ID NOs: 6-20 and 66-67 Is the same gene as the target gene of the microRNA of SEQ ID NO: 1 in Table 1. Further, as a target gene of a nucleic acid consisting of the base sequence represented by any of SEQ ID NOS: 21-40 or 68-92 Can be the same gene as the target gene of the microRNA of SEQ ID NO: 2 in Table 1. Also, as the target gene of the nucleic acid consisting of the base sequence represented by any of SEQ ID NO: 4; In Table 1, the same gene as the target gene of the mitochondrial RNA of SEQ ID NO: 3 can be mentioned, and the target gene of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOS: 53 to 63 is as follows: In Table 1 The same gene as the target gene of the microRNA of SEQ ID NO: 4. In addition, the target gene of a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 64 to 65, 93 is In 1 the same gene as the target gene of the microRNA of SEQ ID NO: 5 can be mentioned.

[表 1]

Figure imgf000017_0001
[table 1]
Figure imgf000017_0001

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(SC9IOZ)60ISA: (98ZSI I) 9ZVSZ31S:aZ9l0Z)696 nd-' (19119)811-1023： (SC9IOZ) 60ISA: (98ZSI I) 9ZVSZ31S: aZ9l0Z) 696 nd- '(19119) 811-1023:

(t 6)蘭 : (98ΠΙ) SSVy: (6 l丄葡: (08ΠΙ)画: (9lit5)0ZV (t 6) Orchid: (98ΠΙ) SSVy: (6 l 丄 葡: (08ΠΙ) Picture: (9lit5) 0ZV

9JIS: (9s A9)g nvvi¾:

Figure imgf000047_0007
(uu)zaim' (ss99 9JIS: (9s A9) g nvvi¾:
Figure imgf000047_0007
(uu) zaim '(ss99

)9V931S: (ZC66) DaSbl： 8ZSS)0fW3WI: (6ZS9)簡 IS: 画: (Z S  ) 9V931S: (ZC66) DaSbl: 8ZSS) 0fW3WI: (6ZS9) Simplified IS: Image: (Z S

)VUIX: (6SZ6) 3Van: ( tSI) lV9ms: (Z5 8)Sino: (06S )IAd(M: ( ) VUIX: (6SZ6) 3Van: (tSI) lV9ms: (Z5 8) Sino: (06S) IAd (M: (

ID (6098UiH : (8Z88)2dMN-' (ZZU)薦: (t666Z)aZZV9- (0m)LU ID (6098UiH: (8Z88) 2dMN- '(ZZU) Recommended: (t666Z) aZZV9- (0m) LU

： (f896Zl)SJVNira: ( 29C)agHNi: (699i9)Sll ed3: (0Z9t8)nV39丄 S: (6  : (F896Zl) SJVNira: (29C) agHNi: (699i9) Sll ed3: (0Z9t8) nV39 丄 S: (6

68S)MdV: (8889S) Id隨: (0 002) 0 00 (H: (8 ) 1 X環: (LZ^DU 68S) MdV: (8889S) Id 隨: (0 002) 0 00 (H: (8) 1 X ring: (LZ ^ DU

。 ( siooz)zaayas-' (1896)舊 d: wnz)o v (ooss uw: (si66 (S89S)aS(i(I r- (69918) (8 SI) W- (【SH)画；): (8i96Z)£1311  . (siooz) zaayas- '(1896) 舊 d: wnz) ov (ooss uw: (si66 (S89S) aS (i (I r- (69918) (8 SI) W- ([SH) picture;); (8i96Z ) £ 1311

IS- ΐ峯  IS- ΐ 峯

LLllLO/LOOZdT/lDd 9 606CS0/800Z OAV [εε挲] [6900] LLllLO / LOOZdT / lDd 9 606CS0 / 800Z OAV [εε 挲] [6900]

W: (S5I98Z)9DSni-' (IIZ Z) 990VVI¾: (288191)麵 Z: ( S) (16 W: (S5I98Z) 9DSni- '(IIZ Z) 990VVI¾: (288191) 麵 Z: (S) (16

6 ISOXSi: 60i dVW: (29816) ε(Π3Λ漏: (9ZZ01) SIVJ : S180U  6 ISOXSi: 60i dVW: (29816) ε (Π3Λ leakage: (9ZZ01) SIVJ: S180U

Ή' (6l0iS) I IdVIO: (ZW)Lkm' (OS 丄 SScLL: (^SIS) 9I IW: (£ ) Ή '(6l0iS) I IdVIO: (ZW) Lkm' (OS 丄 SScLL: (^ SIS) 9I IW: (£)

IDDHV: (6Z9" I画: (t^ZS2)09S0VVIS: (S06Z)VZ : 6H0OHd: ( IDDHV: (6Z9) Image I: (t ^ ZS2) 09S0VVIS: (S06Z) VZ: 6H0OHd: (

lO^ZDSSH V: ( Z Z)蘭 : Π纏: (iS8 ） 9Z謂: (Z996i)0SJJO lO ^ ZDSSH V: (Z Z) Ran: Π Conclusion: (iS8) 9Z so-called: (Z996i) 0SJJO

910： (8S68)8dVIV8: 9W)9l駕: (OS ） MW: (S969S)V腹: (Zmi910: (8S68) 8dVIV8: 9W) 9l 駕: (OS) MW: (S969S) V belly: (Zmi

i : (1996） ΠΙ3Ν: (8U6i,) TI VWi: (16801)320¾03： (09816) 謂:): (ILL  i: (1996) ΠΙ3Ν: (8U6i,) TI VWi: (16801) 320¾03: (09816) so-called :): (ILL

)ZlV3:(【8 SH) 11)1039： aXdH: (8S ） l<fflSSd丄： 請：（896 ) ZlV3: ([8 SH) 11) 1039: aXdH: (8S) l <fflSSd 丄: Contract: (896

00"89600 )ΟΊ: (^86) 9Z10VV -' (008^8) jJ0H3: (S686)6Z£0WI¾: (9 00 "89600) ΟΊ: (^ 86) 9Z10VV-'(008 ^ 8) jJ0H3: (S686) 6Z £ 0WI¾: (9

9ws) 8)¾ ： (m)i a^' (0668 動: a no isaro: (66011  9ws) 8) ¾: (m) i a ^ '(0668 Movement: a no isaro: (66011

) i疆 d: ( mzimd- (L^)mdi ' 8) 腿: (SSIZ)AJ: (omi)v ) i 疆 d: (mzimd- (L ^) mdi '8) Thigh: (SSIZ) AJ: (omi) v

iidiv: (s )sda函:

Figure imgf000048_0001
( ssn 儘: (80 ）固 s : iidiv: (s) sda box:
Figure imgf000048_0001
(ssn 儘: (80) solid s:

m nazwo v ( is) 丄 v: (z ^Dozdmai- (9sz ) 顯 m nazwo v (is) 丄 v: (z ^ Dozdmai- (9sz) 顯

ι-' ( 69) i:ひ ιοεζ) uoiu- (mi) ιχια'· (KHS 3綱 v: (s【86)z丄 13: ι- '(69) i: hi ιοεζ) uoiu- (mi) ιχια' · (KHS Class 3 v: (s [86) z 丄 13:

(6 ） N IN: (6 ） mSV: (86AS)V9JI¾'- (8 9)91V丄 S: ( )圆 d: (16  (6) N IN: (6) mSV: (86AS) V9JI¾'- (8 9) 91V 丄 S: () 圆 d: (16

)91HA3V: (6SSI8)IV88D1S: IS【ZI) ΜΙ : (6nS)VS3(W: (90S I)  ) 91HA3V: (6SSI8) IV88D1S: IS [ZI) ΜΙ: (6nS) VS3 (W: (90S I)

3：

Figure imgf000048_0002
(Z9I0D5I3VO: (101iS)89画丄: ( 689) 2 a ¾: (6SS 3:
Figure imgf000048_0002
(Z9I0D5I3VO: (101iS) 89 Thumbtack: (689) 2 a ¾: (6SS

9) i IV931S ·'

Figure imgf000048_0003
(蕭)醫 M: (OS 9) i IV931S
Figure imgf000048_0003
(蕭) 醫 M: (OS

UVZilOd: (Z^W m- (8Z86) ld33MV: (S 86) ZaS W: UVZilOd: (Z ^ W m- (8Z86) ld33MV: (S 86) ZaS W:

)un- ios) ΠΟΛΟ: SSOI) WDSSS: (6t6 U8 )znd: (扇 9z)蘭  ) un-ios) ΠΟΛΟ: SSOI) WDSSS: (6t6 U8) znd: (Fan 9z) Ran

： ( ) (89901) l : I: ( 858) USI : (9059) 2VID1S: (996)6 : () (89901) l: I: (858) USI: (9059) 2VID1S: (996) 6

03： ( ） d篇: (188)¾8¾3： (m l^l■ ' (98ZIS)88隨: (22501) l删:  03: () d: (188) ¾8¾3: (m l ^ l ■ '(98ZIS) 88 隨: (22501) l 删:

(^m)zzmo- (289g)vsddNi■ ' ( ) mm■ (S6ig)【XIL: (OSSOD  (^ m) zzmo- (289g) vsddNi ■ '() mm ■ (S6ig) [XIL: (OSSOD

OS： (UC6SI) 0ZW3WI- (8Z I \ I) 謂 d: (0995) JVSd： dlWV: (9Ci668) OS: (UC6SI) 0ZW3WI- (8Z I \ I) So-called d: (0995) JVSd: dlWV: (9Ci668)

UJO0lCl:(ISZ9) S : 911I)S丄 GfW: (65901)2(19903- ( Ι)Ζ娜: (9  UJO0lCl: (ISZ9) S: 911I) S 丄 GfW: (65901) 2 (19903- (Ι) Ζ 娜: (9

廳 V: (8Z)08V: (l98I)VlHOi: (568^8) 9£iW ii: (Z9Z9S) VS iH: (  廳 V: (8Z) 08V: (l98I) VlHOi: (568 ^ 8) 9 £ iW ii: (Z9Z9S) VS iH: (

I08)ZS30Id:(CS868)82JJO6D: (90ZCI9)902C193Oi: (50^851) 896 IVVIS: (5I08) ZS30Id: (CS868) 82JJO6D: (90ZCI9) 902C193Oi: (50 ^ 851) 896 IVVIS: (5

Figure imgf000048_0004
m - G刚) n删: (
Figure imgf000048_0004
m-G 刚) n 删: (

0S89) S: (099Z) UNDO - (SZISiS SCId: (0S9SS O SSnd: (19W9HS-' (9Z  0S89) S: (099Z) UNDO-(SZISiS SCId: (0S9SS O SSnd: (19W9HS- '(9Z

66)sa丄 az: 9HS)i9iivviM: (zm i d- (z ^)L zdis' n n  66) sa 丄 az: 9HS) i9iivviM: (zm i d- (z ^) L zdis' n n

8) i : (m) i 醒: {iimt) m'- (9siss) ι遍 v: (膽） : (mD 8) i: (m) i Awaken: (iimt) m'- (9siss) ι v v: (膽): (mD

zi m- (mm)is^ nd' (98t"顧: (oson)sw細: ( n届 3Ί: ( zi m- (mm) is ^ nd '(98t "ref: (oson) sw fine: (n 3Ί: (

eossz)aeiiH)i: (SSI98Z)SV VCIS:

Figure imgf000048_0005
(s s 薦: (sss6)t eossz) aeiiH) i: (SSI98Z) SV VCIS:
Figure imgf000048_0005
(ss recommendation: (sss6) t

IZ- T拏  IZ- T 拏

LLllLO/LOOZdT/lDd 606CS0/800Z OAV //:/ O Ϊ 0L00I>d 6060800AV 8 LLllLO / LOOZdT / lDd 606CS0 / 800Z OAV //: / O Ϊ 0L00I> d 6060800AV 8

Figure imgf000049_0002
Figure imgf000049_0001
Figure imgf000049_0002
Figure imgf000049_0001

Figure imgf000050_0001
Figure imgf000050_0001

OAV/7: z-ZJ0/,0sfc1>d 09 甲OAV / 7: z-ZJ0 /, 0sfc1> d 09

O) ( (U8)目函90H (S uS6603ddSLn9::  O) ((U8) Box 90H (S uS6603ddSLn9 ::

()さ£誦 (asn賺86j震謹ila：:;： () Sa 誦 誦 (asnj86j 謹: ila ::;:

) ()dvIS JHSSS¾:: ) () dvIS JHSSS¾ ::

2 ()H ()¾3S83¾6i6la藝0301V:： ) (舊))函匪sn〕zsl Hi-9osv::  2 () H () ¾3S83¾6i6la 藝 0301V ::) (舊)) Box sn] zsl Hi-9osv ::

(U) ()SSIs13V62ΙaSNa ΠΛn::::  (U) () SSIs13V62ΙaSNa ΠΛn ::::

) ( ( () ())き8sSAA5κVSs8zn3zSHOzεη9 IId：·:::- ()舊 ()§讓〕 d嘆 s Ίss330 aSI laiー； ) ((() ()) 8sSAA5κVSs8zn3zSHOzεη9 IId: · :::-() 舊 () § 讓] d lament s Ίss330 aSI lai ー;

()) £6ss εζ13a£IWS·;:-

Figure imgf000051_0001
() VΓ8as-Π9Ks9≤HiN:.:· .()) £ 6ss εζ13a £ IWS ·;:-
Figure imgf000051_0001
() VΓ8as-Π9Ks9≤HiN:.:

δ)() Η a函 9s as謹βS Ή1ilI3090_:::;  δ) () Η a box 9s as 謹 βS Ή1ilI3090 _ :::;

()舊 (9S SN8ー氧8oois szaGaS£Ha6viwwr8 n:-: )) ( () ( GOHngsszlsd nsS5sdi3S9S1:::一; () 舊 (9S SN8-oxy8oois szaGaS £ Ha6viwwr8 n:-:)) (() (GOHngsszlsd nsS5sdi3S9S1 ::: One;

) () (O6U3S35變編I z9i686av3vαΗΊ>ρ S:：;  ) () (O6U3S35 變 I z9i686av3vαΗΊ> ρ S ::;

() (s£ (ii3aNS) Oa9so6SSJiSUHS69 Ί::() (s £ (ii3aNS) Oa9so6SSJiSUHS69 Ί ::

§ ()dwvr (s 0s65£06s3snNZvld mvavv: § () dwvr (s 0s65 £ 06s3snNZvld mvavv:

s ( (0)2 S s92iso s ddlHvdlASOA1H A::::  s ((0) 2 S s92iso s ddlHvdlASOA1H A ::::

) l ())6議 (l0s (ss9z3wsv¾il3s§N8la：^::; ) l ()) 6 (l0s (ss9z3wsv¾il3s§N8la: ^ ::;

) ( STSJXs) IdaVs 96 si::"  ) (STSJXs) IdaVs 96 si :: "

()6 sss60S (H。3ddHnsHa:.::. § (霄 liiw)6S≤ ()A¾ iu (ss (n59s5llnu¾ds 5d一：::: ¾星9ε2

Figure imgf000052_0001
() 6 sss60S (H.3ddHnsHa:. ::. § (霄 liiw) 6S≤ () A¾ iu (ss (n59s5llnu¾ds 5d one :::: ¾ star 9ε2
Figure imgf000052_0001

6602) :歸 ) 腿 Ή: ( m) UJJ09D-' (iS6 I) IS: (iS6 )V 6602): 歸) Thigh Ή: (m) UJJ09D- '(iS6 I) IS: (iS6) V

zmr- (wiL^n ^- 6) d爾: wm)£mmv 9u) nzm (ss zmr- (wiL ^ n ^-6) d 爾: wm) £ mmv 9u) nzm (ss

mi) ussDis-' (ion ） 8丄 093:

Figure imgf000052_0002
muu mi) ussDis- '(ion) 8 丄 093:
Figure imgf000052_0002
muu

) \3QUH' (8ZS8)Oda: (ZS 6) W: (S 02) VHda: ( i)<n i人 S: (漏） 3  ) \ 3QUH '(8ZS8) Oda: (ZS 6) W: (S 02) VHda: (i) <n i people S: (Leakage) 3

51Ν'· (CSOCZ) U3J0CI: ( S£S5)V0CW3Ki: (88 SO I删: (98812^) 991-1093： 51Ν '· (CSOCZ) U3J0CI: (S £ S5) V0CW3Ki: (88 SO I 删: (98812 ^) 991-1093:

(8"I8) IVinOD: (89Z9 60Χ : ( i£S8)8H&Vd: (i9I) IdS : (S009)9VHH (8 "I8) IVinOD: (89Z9 60Χ: (i £ S8) 8H & Vd: (i9I) IdS: (S009) 9VHH

： (098)ΖΧΚΠΗ·' (mZ)漏 6dSH: (OIS ) : (56901) 933^1： (CII9II)tJX : (098) ΖΧΚΠΗ · '(mZ) leakage 6dSH: (OIS): (56901) 933 ^ 1: (CII9II) tJX

Oil: (S986）nW3M: (8 9)SSMS: (8992^)691-1090： (i ZD^li ' (68 Oil: (S986) nW3M: (8 9) SSMS: (8992 ^) 691-1090: (i ZD ^ li '(68

zi) iM' (mzzDimn^' (969s)謂 sd: (8i ） ZV&Q- VIH: (68os ¾d: ( zi) iM '(mzzDimn ^' (969s) so-called sd: (8i) ZV & Q- VIH: (68os ¾d: (

H901) I V: ( : (6S ) S翻:

Figure imgf000052_0003
(け 0I)NS( : (9595 H901) IV: (: (6S) S Translation:
Figure imgf000052_0003
(Ke 0I) NS (: (9595

SI) m - (8S99S)6SWIM: ( OlOI)OIWIM: (99C8)H HUSm: (9S8SS)画 (SI) m-(8S99S) 6SWIM: (OlOI) OIWIM: (99C8) H HUSm: (9S8SS)

丄： (9061)圆: ( 9)9纖: ) I'S讓 ε- 9)381-1093： (0iZ9S) 2303： iU8Snd: ( ΙΠ) X: (9699) TOO 丄: (9061) 圆: (9) 9 纖:) I'S 讓 ε- 9) 381-1093: (0iZ9S) 2303: iU8Snd: (ΙΠ) X: (9699) TOO

dS: (6ZSS91)61Nia: (^66 LW3: (9I6Z)隨 3: (H )翻: (Z9Z9ZUINV dS: (6ZSS91) 61Nia: (^ 66 LW3: (9I6Z) 隨 3: (H) Translation: (Z9Z9ZUINV

dS丄: (Z99) I續 8: (Z2CSSI)Z^8 l30i: ( ) IdSHd: m ) Id画: (98S dS 丄: (Z99) I 續 8: (Z2CSSI) Z ^ 8 l30i: () IdSHd: m) Id image: (98S

9 ins:

Figure imgf000052_0004
( 9 ins:
Figure imgf000052_0004
(

9Z80I) WJOSD: (^801)21^93： (QUZ) IXSaN-' ( 18)¥»3- OS OaiJSD'  9Z80I) WJOSD: (^ 801) 21 ^ 93: (QUZ) IXSaN- '(18) ¥ »3- OS OaiJSD'

(SHS)V93ad: (8Z96i) ISHS: (OZSIS)smn: (89A8 1) WI OWS (U£S5)0 (SHS) V93ad: (8Z96i) ISHS: (OZSIS) smn: (89A8 1) WI OWS (U £ S5) 0

83圆： (SW9Z)ZNim: (36ίΊ) IVMD: (SSSfrSl 9S nd: (8£g£2) SliHd 83 圆 ： (SW9Z) ZNim: (36ίΊ) IVMD: (SSSfrSl 9S nd: (8 £ g £ 2) SliHd

： (SZU tJdV: (90£S2)91SDV: ( 906) 906:)ΟΊ: (9SSiS)V9VW3S: (C91t)3 : (SZU tJdV: (90 £ S2) 91SDV: (906) 906:) ΟΊ: (9SSiS) V9VW3S: (C91t) 3

on- (Lzzm)zi a' ( 6) ： (so6 ) wissis: (01 ) on- (Lzzm) zi a '(6): (so6) wissis: (01)

ίχπ-' usiis)卜 s丄 (mm) omzni- ^ ^D um- am) ε醫 ίχπ- 'usiis) 卜 s 丄 (mm) omzni- ^ ^ D um- am) ε 醫

： (6lil)腿 a: ½2S6)I層: IS: (ISけ） (f^6 )SV0S:nS: (98 i9) lN^ : (6lil) thigh a: ½2S6) I layer: IS: (IS) (f ^ 6) SV0S: nS: (98 i9) lN ^

Figure imgf000052_0005
666 13: (95099) ΜΜϋ' (896991) 6111118idZiI
Figure imgf000052_0005
666 13: (95099) ΜΜϋ '(896991) 6111118idZiI

¾a: (讓 lusj: (mz) zvaii: (8itsn)aw3: is) is雇: m) iv雷 d  ¾a: (讓 lusj: (mz) zvaii: (8itsn) aw3: is) is hired: m) iv thunder d

：

Figure imgf000052_0006
os9)svms: os9)2d¾s: (889εεΐ) m-:
Figure imgf000052_0006
os9) svms: os9) 2d¾s: (889εεΐ) m-

(6Z695) 6瞧 d: (66Z0I)霞 VW: (iS06)VSVW3S: (80I)Z人：) (簡 1) (6Z695) 6 瞧 d: (66Z0I) 霞 VW: (iS06) VSVW3S: (80I) Z people :) (simplified 1)

siod-(zino )o9S8snii-' (i6t )MHV: (98 c) i 11 i 99dZ;isa-' (mzoz) siod- (zino) o9S8snii- '(i6t) MHV: (98 c) i 11 i 99dZ; isa-' (mzoz)

3VN3¾: (28ZIl)a IVOW: 1008) 1808) ZUIVVIM: IS 3VN3¾: (28ZIl) a IVOW: 1008) 1808) ZUIVVIM: IS

3)95Π1Πίΐ: ( 6)環 Ί: (mi)m' 議 Od: )0Ί  3) 95Π1Πίΐ: (6) Ring Ί: (mi) m 'Meeting Od:) 0Ί

:(6t^)iaV3:O8 S)0SI;I : (6I8S2) ]：): (U 95C) 2082230^： (9iUS  : (6t ^) iaV3: O8 S) 0SI; I: (6I8S2)] :): (U 95C) 2082230 ^: (9iUS

) (6Ζ6991) 8969Ζ33Μ'· (0 : (U90I) 9ΜΠ(Μ: (S^S )S18il ) (6Ζ6991) 8969Ζ33Μ '· (0: (U90I) 9ΜΠ (Μ: (S ^ S) S18il

CM: (66 ） : (mm)zi} - (umi)zui^' (106) (S9i¾  CM: (66): (mm) zi}-(umi) zui ^ '(106) (S9i¾

) lavas: (0i s)Zii3dd: (si8zsi)9dVHi: (868 Z) HO¾: (6902) saaa: (woけ ) lavas: (0i s) Zii3dd: (si8zsi) 9dVHi: (868 Z) HO¾: (6902) saaa: (wo

LLllLO/LOOZdT/lDd 1-9 606CS0/800Z OAV LLllLO / LOOZdT / lDd 1-9 606CS0 / 800Z OAV

Figure imgf000053_0001
Figure imgf000053_0001

〔s寸 90 [6ε挲] [9900] [S dimension 90 [6ε 挲] [9900]

Ull) 13 : ( 89)ΝΊ3ν丄：（6WS 麵 Vd: (StS) S30<IV: (6SSS)VSH1H: Ull) 13: (89) ΝΊ3ν 丄 ： (6WS 麵 Vd: (StS) S30 <IV: (6SSS) VSH1H:

89 ) IWV3N- (Z6S9)dH(lS: {ΙΖΙ\)Ι ΊΆ· (6I99)aiH2iJj: (699A9) OZJVOfflV: ( 89) IWV3N- (Z6S9) dH (lS: (ΙΖΙ \) Ι ΊΆ ・ (6I99) aiH2iJj: (699A9) OZJVOfflV: (

666S) 666S3(n: (10816)8画 (2^62 ) (S69SS)d Wf: (n^Z 666S) 666S3 (n: (10816) 8 strokes (2 ^ 62) (S69SS) d Wf: (n ^ Z

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9) 1WIIS- (S90A9)0IVN¾HD: (0581)800： 【【S) HVdSH: ( ) [VMO- 99)8dINg: (16ZS)HHJ1J: (CZ866S) IE89tni: 1WV0V: (88^1)2(1313： 9) 1WIIS- (S90A9) 0IVN¾HD: (0581) 800: [[S) HVdSH: () [VMO- 99) 8dINg: (16ZS) HHJ1J: (CZ866S) IE89tni: 1WV0V: (88 ^ 1) 2 (1313:

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η· (Ot AVA: (8Z)09V: (1919S) HHHVe: ( 0699)29198HS-' (588^8)213101 η (Ot AVA: (8Z) 09V: (1919S) HHHVe: (0699) 29198HS- '(588 ^ 8) 213101

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LLllLO/LOOZdT/lDd 99 606CS0/800Z OAV 表 1一 42 LLllLO / LOOZdT / lDd 99 606CS0 / 800Z OAV Table 1 1 42

r;RBM12(10137);C20orf4(25980)； DRG3 (57446)； C20orfl 17(140710)； RPN 2(6185)； PTPRT(11122)； SERINC3(10955)； KCNS1 (3787)； WFDC5 (149708) ;W FDC12 (128488)； SLC13A3 (64849)； SULF2 (55959)； ARFGEF2 (10564)； SPATA2 (9825)； ZNF313 (55905)； PTPN1 (5770)； KCNG1 (3755)； ATP9A (10079)； ZFP64 (55734)； RAB22A(57403)； CDH4C1002)； GTPBP5(26164)； PRIC285 (85441)； M YT1 (4661) ;CHRNA (1137) ;PARD6B(84612)； KIAA1219(57148)； ABCC13(150 R; RBM12 (10137); C20orf4 (25980); DRG3 (57446); C20orfl 17 (140710); RPN 2 (6185); PTPRT (11122); SERINC3 (10955); KCNS1 (3787); WFDC5 (149708); W FDC12 (128488); SLC13A3 (64849); SULF2 (55959); ARFGEF2 (10564); SPATA2 (9825); ZNF313 (55905); PTPN1 (5770); KCNG1 (3755); ATP9A (10079); ZFP64 (55734); RAB22A (57403); CDH4C1002); GTPBP5 (26164); PRIC285 (85441); M YT1 (4661); CHRNA (1137); PARD6B (84612); KIAA1219 (57148); ABCC13 (150

000) ;ADA TS5 (11096)； KRTAP26-1 (388818) ;SLC5A3 (6526)； HLCS (3141)； B R D1 (54014)； WRB(7485)； DSCAM (1826) ;ZNF295 (49854)； ABCG1 (9619)； KIA AO 179 (23076)； PDXK (8566)； LRRC3 (81543)； KRTAP 10-12 (386685)； COL6A2 ( 1292)； CECR1 (51816)； DGCR20993)； DGCR14(8220)； LOCI 28977 (128977)； H TF9C(27037)； PCQAP (51586)； PIK4CA(5297) ;CRKL(1399)； MAPK1 (5594)； LO C51233 (51233)； UPB1 (51733) ;C22orfl3 (83606)； MN1 (4330)； FLJ33814 (15 0275)； SLC5A1 (6523)； SOX10 (6663)； KDELR301015)； RP5-1104E15.5 (5447000); ADA TS5 (11096); KRTAP26-1 (388818); SLC5A3 (6526); HLCS (3141); BR D1 (54014); WRB (7485); DSCAM (1826); ZNF295 (49854); ABCG1 (9619) ; KIA AO 179 (23076); PDXK (8566); LRRC3 (81543); KRTAP 10-12 (386685); COL6A2 (1292); CECR1 (51816); DGCR20993); DGCR14 (8220); LOCI 28977 (128977) HTF9C (27037); PCQAP (51586); PIK4CA (5297); CRKL (1399); MAPK1 (5594); LO C51233 (51233); UPB1 (51733); C22orfl3 (83606); MN1 (4330); FLJ33814 ( 15 0275); SLC5A1 (6523); SOX10 (6663); KDELR301015); RP5-1104E15.5 (5447)

1)； TNRC6BC23112)； MKL1 (57591)； RBX1 (9978)； L3MBTL2 (83746)； ZC3H7B(2 3264)； ACO2(50)： POL腿（171568)； P0LDIP3 (84271)； ARFGAP3 (26286)； FBL N1 (2192)； FLJ27365(400931)； FLJ41993 (400935)； ADM2 (79924)； AP0L6(80 830)： TR匿（11078)； P2RY8 (286530)； ZBED1 (9189)； APXL (357)； FAM9C(17 1484)； GPM6B(2824)； TMEM27 (57393)； SH3KBP1 (30011)； RPS6KA3 (6197)； P0 LA(5422)；画（257397)； PRRG1 (5638)； DP (4693)； CXor f 36 (79742)； INE1 (8552);ZNF21 (7569)； PIM2O 1040)； SMCX(8242)； IQSEC2 (23096)； SMC1L1 ( 8243)； TMEM28(27112)； TAF1 (6872) ;OGT(8473)； RP11-130N24.1 (340533)； ATRX(546)； PGK1 (5230)； TAF9B (51616)； P0F1BC79983)； DIAPH2 (1730)； TSP AN6 (7105)； 0RF4L2C9643)； MGC39900(286527)： RP1卜 321G1.1 (54885)； RP 13-383K5.1(55285)； NXT2C55916)； TRPC5 (7224)； ZCCHC16 (340595)； HTR2C (3358)； UPF3B(65109)； SUHW3 (55609)：删 X (27316)； LOC401620(401620)； MECP2 4204)； DNASE1L1 (1774)； UBL4A(8266)； GAB3 (139716)； IL9R(3581)； AFF2 (2334) ;ZFY (7544)； CD24C934)； SMCYC8284)； 1); TNRC6BC23112); MKL1 (57591); RBX1 (9978); L3MBTL2 (83746); ZC3H7B (2 3264); ACO2 (50): POL thigh (171568); P0LDIP3 (84271); ARFGAP3 (26286); FBL N1 FLJ27365 (400931); FLJ41993 (400935); ADM2 (79924); AP0L6 (80 830): TR concealment (11078); P2RY8 (286530); ZBED1 (9189); APXL (357); FAM9C (17 1484) GPM6B (2824); TMEM27 (57393); SH3KBP1 (30011); RPS6KA3 (6197); P0 LA (5422); Drawing (257397); PRRG1 (5638); DP (4693); CXor f 36 (79742); INE1 (8552); ZNF21 (7569); PIM2O 1040); SMCX (8242); IQSEC2 (23096); SMC1L1 (8243); TMEM28 (27112); TAF1 (6872); OGT (8473); RP11-130N24.1 ( ATRX (546); PGK1 (5230); TAF9B (51616); P0F1BC79983); DIAPH2 (1730); TSP AN6 (7105); 0RF4L2C9643); MGC39900 (286527): RP1 卜 321G1.1 (54885); RP 13-383K5.1 (55285); NXT2C55916); TRPC5 (7224); ZCCHC16 (340595); HTR2C (3358); UPF3B (65109); SUHW3 (55609): 删 X (27316); LOC401620 (401620); MECP2 4204 DNASE1L1 (1774) UBL4A (8266); GAB3 (139716); IL9R (3581); AFF2 (2334); ZFY (7544); CD24C934); SMCYC8284);

[0069] 本発明において、 ADD3遺伝子とは、配列番号 206で表される塩基配列に含まれ るヒト adducin3 (g匪 ma)遺伝子、およびそれに対応する他生物種の相同遺伝子であ るォーソログをいう。また、 CBFB遺伝子とは、配列番号 207で表される塩基配列に 含まれるヒト core-binding factor, beta subnit (CBFb)遺伝子、およびそれに対する他 生物種の相同遺伝子であるォーソログをいう。 [0069] In the present invention, the ADD3 gene refers to a human adducin3 (g 匪 ma) gene contained in the base sequence represented by SEQ ID NO: 206, and an ortholog that is a homologous gene of another species corresponding thereto. . The CBFB gene refers to a human core-binding factor, beta subnit (CBFb) gene contained in the base sequence represented by SEQ ID NO: 207, and orthologs that are homologous genes of other species.

[0070] 本発明のマイクロ RNAの標的遺伝子の発現を抑制する物質とは、該マイクロ RNA の標的遺伝子の mRNAの発現を抑制する活性を有する物質であればいかなる物質 であってもよいが、核酸が好ましぐ siRNAが特に好ましい。なお、発現を抑制する活 性とは、（l ) mRNAの翻訳を抑制させる活性、および（2) mRNAを切断あるいは分解 することによって、結果として mRNAから翻訳される蛋白質の量を減少させる活性をい う。 ADD3遺伝子の発現を抑制する物質の具体的な例として、 ADD3遺伝子に対す る siRNAをあげることができ、より好ましくは、配列番号 202〜203のいずれかで表さ れる塩基配列を標的配列とする siRNAをあげることができる。 CBFB遺伝子の発現を 抑制する物質の具体的な例として、 CBFB遺伝子に対する siRNAをあげること力 Sでき 、より好ましくは、配列番号 204〜205のいずれかで表される塩基配列を標的配列と する siRNAをあげることができる。 [0070] The substance that suppresses the expression of the target gene of the microRNA of the present invention may be any substance as long as it has an activity of suppressing the expression of the mRNA of the target gene of the microRNA. SiRNA is particularly preferred. In addition, activity to suppress expression Sex refers to (l) the activity of suppressing the translation of mRNA, and (2) the activity of cleaving or degrading the mRNA, resulting in a decrease in the amount of protein translated from the mRNA. As a specific example of a substance that suppresses the expression of the ADD3 gene, siRNA for the ADD3 gene can be exemplified, and more preferably, the base sequence represented by any one of SEQ ID NOs: 202 to 203 is used as the target sequence. siRNA can be raised. As a specific example of a substance that suppresses the expression of the CBFB gene, siRNA against the CBFB gene can be raised, and more preferably, the siRNA having the base sequence represented by any of SEQ ID NOs: 204 to 205 as a target sequence Can give.

[0071] 本発明において、間葉系幹細胞の増殖および/または分化の異常に起因する疾 患としては、間葉系幹細胞の増殖および/または分化の異常に起因する疾患であれ ばとくに制限はないが、具体的には、癌及び、骨形成不全症、軟骨形成不全症、糖 尿病等をあげることができる。  [0071] In the present invention, the disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells is not particularly limited as long as it is a disease caused by abnormal proliferation and / or differentiation of mesenchymal stem cells. However, specific examples include cancer, osteogenesis dysplasia, chondrogenesis dysplasia, and glucoseuria.

以下、本発明を具体的に説明する。  The present invention will be specifically described below.

1.聞蔞系榦細朐で発現するマイクロ RNAおよびマイクロ RNA前駆体の同定  1. Identification of microRNAs and microRNA precursors expressed in the hearing system

( 1 1 )間葉系幹細胞の取得および培養  (1 1) Acquisition and culture of mesenchymal stem cells

骨髄から間葉系幹細胞を取得する方法としては、安全かつ効率的に取得される方 法であれば特に限定されないが、例えば S. E. Haynesworth et al. Bone, 13， 81 (199 2)に記載の方法があげられる。  The method for obtaining mesenchymal stem cells from the bone marrow is not particularly limited as long as it is a safe and efficient method. For example, the method described in SE Haynesworth et al. Bone, 13, 81 (199 2) Can be given.

[0072] 胸骨または腸骨において、骨髄穿刺を行う場所の皮膚を消毒し、特に骨膜下を十 分に局所麻酔する。骨髄穿刺針の内筒を抜き、 5,000 unitsのへノ リンを入れた 10 ml 注射器を装着して必要量、平均的には 10 ml〜20 mlの骨髄液を吸引する。骨髄穿刺 針を取り外し、 10分間程度圧迫止血する。取得した骨髄液を 1 ,000 X gで遠心分離し て骨髄細胞を回収した後、該骨髄細胞をリン酸緩衝液（phosphate-buffered saline) (P BS)で洗浄する。遠心分離'洗浄を 2回繰り返した後、骨髄細胞を 10%のゥシ胎仔血 清（FBS)を含む a -MEM ( a -modified MEM), DMEM (Dulbecco's modified MEM), I MDM (Isocove's modified Dulbecco's medium)等の細胞培養用培地に浮遊させること により骨髄細胞液を得る。骨髄細胞液から間葉系幹細胞を単離する方法としては、 骨髄細胞液中に混在する他の細胞、例えば血球系細胞、造血幹細胞、血管幹細胞 、泉維芽細胞等を除去することができれば特に限定されないが、例えば M. F. Pitten ger et al. Science, 284， 143-147 (1999)に記載の方法があげられる。骨髄細胞液を 密度 1.073 g/mlの Percollに重層した後、 1， 100 X gで 30分間遠心分離し、界面の細胞 を間葉系幹細胞として単離することができる。また、骨髄細胞液に 10 X PBSを加えて 9 /10に希釈した Percollを同容量加えて混合した後に、 20,000 X gで 30分間遠心分離し 、密度 1·075〜1·060の画分の細胞を間葉系幹細胞として単離することもできる。 [0072] In the sternum or iliac, disinfect the skin where the bone marrow puncture is performed, and in particular, sufficiently anesthetize the subperiosteum. Remove the inner tube of the bone marrow puncture needle and attach a 10 ml syringe containing 5,000 units of heline to aspirate the required amount, typically 10 ml to 20 ml of bone marrow. Remove the bone marrow puncture needle and press and stop for about 10 minutes. The obtained bone marrow fluid is centrifuged at 1,000 X g to collect bone marrow cells, and then the bone marrow cells are washed with phosphate-buffered saline (PBS). After centrifugation and washing twice, bone marrow cells containing 10% urine fetal serum (FBS) a -MEM (a -modified MEM), DMEM (Dulbecco's modified MEM), I MDM (Isocove's modified Dulbecco's A bone marrow cell solution is obtained by suspending in a cell culture medium such as medium). As a method for isolating mesenchymal stem cells from bone marrow cell fluid, other cells mixed in the bone marrow cell fluid such as blood cells, hematopoietic stem cells, hemangioblasts The fountain fibroblasts can be removed without particular limitation, and examples thereof include the method described in MF Pittenger et al. Science, 284, 143-147 (1999). The bone marrow cell solution can be layered on Percoll with a density of 1.073 g / ml, then centrifuged at 1,100 X g for 30 minutes to isolate the cells at the interface as mesenchymal stem cells. Also, add 10X PBS to the bone marrow cell solution and mix with Percoll diluted to 9/10, add the same volume and mix, then centrifuge for 30 minutes at 20,000 X g, and collect the fraction of density 1.075-1060 Cells can also be isolated as mesenchymal stem cells.

[0073] また、ヒトの骨髄に由来する間葉系幹細胞は、 Cambrex社、タカラバイオ社より購入 することあでさる。 [0073] Further, mesenchymal stem cells derived from human bone marrow can be purchased from Cambrex and Takara Bio.

臍帯から間葉系幹細胞を取得する方法としては、効率的に取得される方法であれ ば特に限定されないが、例えば Stem Cells, 21， 105-110 (2003)に記載の方法があげ られる。臍帯静脈の両端に力ニューレを揷入し、適当な緩衝液、例えば、 EBSS(Earle' s balanced salt solution)で洗浄する。蛋白質分解酵素、例えば、 0.1%コラゲナーゼを 含む 199培地に抗生物質を添加して血管に注入し、 4〜40 °C、好ましくは 37 °Cで、 1 〜60分間インキュベートする。血管を EBSSで洗浄し、臍帯を軽くマッサージした後、 内皮細胞および内皮下層細胞の懸濁液を回収する。該懸濁液を 600 X gで 10分間遠 心分離し、得られた細胞を、例えば、低濃度のグルコースを含む DMEM培地（DMEM - LG、 Gibco)に、 20 mM HEPES、 100 units/mlペニシリン、 100 μ g/mlストレプトマイ シン、 2 mM L -グルタミン、 1 mMピルビン酸ナトリウムおよび 10% FBSを加えた培地に 懸濁する。細胞密度を、 102〜106個ん m2として培養フラスコに接種し、 37 °C、 5% CO の条件下で培養する。培地を 1〜7日毎に交換し、；!〜 3週間培養を継続することによ り、間葉系幹細胞を取得することができる。 The method for obtaining mesenchymal stem cells from the umbilical cord is not particularly limited as long as it is an efficient method, and examples thereof include the method described in Stem Cells, 21, 105-110 (2003). A force neulet is inserted into both ends of the umbilical vein and washed with an appropriate buffer, for example, EBSS (Earle's balanced salt solution). Antibiotics are added to a 199 medium containing a proteolytic enzyme, for example, 0.1% collagenase, injected into a blood vessel, and incubated at 4 to 40 ° C, preferably 37 ° C for 1 to 60 minutes. After washing the blood vessels with EBSS and lightly massaging the umbilical cord, the suspension of endothelial cells and subendothelial cells is collected. The suspension was centrifuged at 600 × g for 10 minutes, and the obtained cells were placed in, for example, DMEM medium (DMEM-LG, Gibco) containing low concentration of glucose, 20 mM HEPES, 100 units / ml penicillin. Suspend in medium supplemented with 100 μg / ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 10% FBS. The culture flask is inoculated at a cell density of 10 2 to 10 6 m 2 and cultured under conditions of 37 ° C. and 5% CO 2. Mesenchymal stem cells can be obtained by changing the medium every 1 to 7 days and continuing the culture for! ~ 3 weeks.

[0074] 子宮内膜から間葉系幹細胞を取得する方法としては、安全かつ効率的に取得され る方法であれば特に限定されないが、例えば、 Am. J. Pathol., 163， 2259-2269 (200 3)に記載された方法があげられる。外科手術により摘出されたヒト子宮内膜組織を細 切し、細胞を培養可能な培地、好ましくは α _ΜΕΜ、 DMEM, IMDM等に 1〜20%の 動物由来の血清、好ましくは 5〜10%の FBSを加えた培地で培養する。培地にはぺニ シリンやストレプトマイシン等の抗生物質を加えても良い。さらに、細胞の分離を良く するために、 3型コラゲナーゼ等のコラーゲン分解酵素およびデォキシリボヌクレア一 ゼ I等の DNA分解酵素を培地に添加し、 20〜40 °C、好ましくは 37 °Cの条件で、 10分 間〜 5時間、好ましくは 1時間、緩やかに振盪する。個々の子宮内膜腺を顕微鏡で観 察しながら分離し、適当な培養容器、例えば 24-ゥエル培養皿を用いて 37 °C、 5% C 0の条件下で培養することにより、間葉系幹細胞を取得することができる。 [0074] The method for obtaining mesenchymal stem cells from the endometrium is not particularly limited as long as it is a safe and efficient method. For example, Am. J. Pathol., 163, 2259-2269 ( 200 3). Human endometrial tissue excised by surgery is cut into small pieces and cultured in a culture medium, preferably α_ΜΕΜ, DMEM, IMDM, etc. 1-20% animal-derived serum, preferably 5-10% Incubate in medium supplemented with FBS. Antibiotics such as penicillin and streptomycin may be added to the medium. In addition, collagen-degrading enzymes such as type 3 collagenase and deoxyribonuclease Add DNA-degrading enzyme such as zase I to the medium and gently shake at 20-40 ° C, preferably 37 ° C for 10 minutes to 5 hours, preferably 1 hour. Individual endometrial glands can be isolated while observing under a microscope and cultured in an appropriate culture vessel such as 24-well culture dish at 37 ° C and 5% C 0 to obtain mesenchymal stem cells. Can be obtained.

[0075] 歯、歯胚、歯周辺組織から間葉系幹細胞を取得する方法としては、安全かつ効率 的に取得される方法であれば特に限定されないが、例えば Lancet, 364， 149-155 (2 004)、 Proc. Natl. Acad. Sci. USA, 97， 13625-13630 (2000)に記載の方法があげられる 。ヒトの歯としては、乳歯、または切歯、犬歯、小臼歯、大臼歯等の永久歯のいずれ でも良い。例えば、抜歯した第三大臼歯 (親知らず）の歯根の表面から歯周靭帯 (per iodontal ligament)を慎重に分離し、コラゲナーゼ、トリプシン、プロナーゼ、ェラスタ ーゼ、デイスパーゼ、ヒアルロニダーゼ等の蛋白分解酵素を用いて、 37 °Cで 1時間消 化反応を行う。ストレイナー、メッシュ、フィルタ一等を用いて、組織残渣を除くことによ り間葉系幹細胞を取得することができる。また、抜歯した第三大臼歯 (親知らず）の表 面を PBS等で洗浄した後、セメント質とエナメル質の結合部を切断して髄質を露出さ せ、歯冠および歯根から歯髄組織を慎重に分離し、上記と同様の方法で蛋白分解 酵素処理した後、組織残渣を除くことにより間葉系幹細胞を取得することもできる。  [0075] The method for obtaining mesenchymal stem cells from teeth, tooth embryos, and tissues around teeth is not particularly limited as long as it is a method that can be obtained safely and efficiently. For example, Lancet, 364, 149-155 (2 004), Proc. Natl. Acad. Sci. USA, 97, 13625-13630 (2000). Human teeth may be milk teeth or permanent teeth such as incisors, canines, premolars, and molars. For example, the periodontal ligament is carefully separated from the surface of the extracted third molar (wisdom tooth) root, and proteolytic enzymes such as collagenase, trypsin, pronase, elastase, dispase, and hyaluronidase are used. Perform the quenching reaction at 37 ° C for 1 hour. Mesenchymal stem cells can be obtained by removing tissue residues using a strainer, mesh, filter, etc. In addition, the surface of the extracted third molar (wisdom tooth) is washed with PBS, and then the cementum and enamel joints are cut to expose the medulla, and the pulp tissue is carefully removed from the crown and root. Mesenchymal stem cells can be obtained by separating and treating with proteolytic enzyme in the same manner as described above, and then removing the tissue residue.

[0076] また、上記以外の間葉系幹細胞単離方法として、間葉系幹細胞に発現する表面抗 原、間葉系幹細胞特異的な遺伝子のプロモーターおよびェンノヽンサ一を持つレポ一 ターベクターを用いて間葉系幹細胞を単離する方法があげられる。具体的には、 AC [0076] Further, as a method for isolating mesenchymal stem cells other than the above, a reporter vector having a surface antigen expressed in mesenchymal stem cells, a promoter of a gene specific to mesenchymal stem cells and an enzyme is used. And a method for isolating mesenchymal stem cells. Specifically, AC

133抗原を用いて幹細胞単離を単離する方法（US6468794)、 Sox遺伝子（US2002/01 35539)、 Nestin遺伝子または Musashi遺伝子 (特開 2002-034580)のプロモーターおよ びェンハンサーを持つレポーターベクターを用いて間葉系幹細胞を単離する方法等 力 fcげられる。 133 Method for isolating stem cell isolation using antigen (US6468794), Sox gene (US2002 / 01 35539), Nestin gene or Musashi gene (JP 2002-034580) promoter and enhancer reporter vector For example, a method for isolating mesenchymal stem cells.

[0077] また、 H0eChst33342の細胞外排出能を指標にした FACS分画法を用いてサイドポピ ユレーシヨン（side population) (SP)中に幹細胞を濃縮させる方法 [Journal of Experim ental Medicine, 183， 1797-806 (1996)]を用いて幹細胞を分離することもできる。また 、 SH2陽性， SH4陽性， CD29陽性， CD44陽性， CD71陽性， CD90陽性， CD 106陽性， CD 120a陽性， CD124陽性， CD14陰性， CD34陰性，および CD45陰性の細胞を間葉 系幹細胞として、セルソーターや磁気ビーズを用いて分離する方法 [Science, 284, 14 3-147 (1999)]を用いることもできる。 [0077] In addition, H 0eC Saidopopi Yureshiyon (side population) (SP) method of concentrating the stem cells in using FACS fractionation method was an indicator extracellular Emptying of hst33342 [Journal of Experim ental Medicine, 183, 1797 -806 (1996)] can also be used to isolate stem cells. In addition, cells that are SH2 positive, SH4 positive, CD29 positive, CD44 positive, CD71 positive, CD90 positive, CD 106 positive, CD 120a positive, CD124 positive, CD14 negative, CD34 negative, and CD45 negative are mesenchymal. As a stem cell, a method of separating using a cell sorter or magnetic beads [Science, 284, 14 3-147 (1999)] can also be used.

[0078] 間葉系幹細胞の培養に用いる培地としては、例えば組織培養の技術基礎編 第三 版、朝倉書店 (1996)等に記載された細胞培養用培地があげられるが、ゥシゃヒト等の 血清を 1〜20%添加した α _ΜΕΜ、 DMEM、 IMDM等の細胞培養用培地が好ましい。 培養条件は、細胞が培養可能であればいかなる条件でもよいが、培養温度は 33〜3 7 °Cが好ましぐ 5〜10%の COガスで満たしたインキュベーターで培養することが好ま しい。間葉系幹細胞は、通常の組織培養用のプラスチック製培養皿に接着させて増 殖させることが好ましい。細胞が培養皿一面に増殖する頃、培地を除去して、トリプシ ン EDTA溶液を加えることで細胞を浮遊させる。浮遊させた細胞は、 PBSまたは細胞 培養用の培地で洗浄後、細胞培養用の培地で 2倍から 20倍に希釈して新し!/、培養 皿に播種することで、さらに継代培養することができる。 [0078] Examples of the medium used for culturing mesenchymal stem cells include cell culture media described in, for example, Tissue Culture Technology Fundamentals 3rd Edition, Asakura Shoten (1996), etc. A cell culture medium such as α_ΜΕΜ, DMEM, IMDM and the like to which 1 to 20% of serum is added is preferable. The culture conditions may be any conditions as long as the cells can be cultured, but the culture temperature is preferably 33 to 37 ° C. The culture is preferably performed in an incubator filled with 5 to 10% CO gas. The mesenchymal stem cells are preferably allowed to grow by adhering to a normal tissue culture plastic culture dish. When the cells grow on the entire culture dish, remove the medium and add the trypsin EDTA solution to suspend the cells. Suspended cells are washed with PBS or cell culture medium, diluted to 2 to 20 times with cell culture medium and freshly seeded in a culture dish, and further subcultured. be able to.

(1 2)低分子 RNAとその配列情報の取得  (1 2) Acquisition of small RNA and its sequence information

上記の各種方法で取得した間葉系幹細胞から全 RNAを抽出し、該 RNAを用いて間 葉系幹細胞で発現するマイクロ RNAを含有する低分子 RNAを以下のようにして取得 すること力 Sでさる。  Extracting total RNA from mesenchymal stem cells obtained by the various methods described above, and using the RNA to obtain low molecular weight RNA containing microRNAs expressed in mesenchymal stem cells as follows: Monkey.

[0079] 低分子 RNAの取得法としては、具体的には、ジーンズ アンド デベロップメント（Ge nes & Development) 15, 188-200 (2000)に記載の方法に準じて、 15%ポリアクリルアミ ドゲル電気泳動による低分子 RNAの分離及び切り出し、 5 '末端脱リン酸化、 3 'ーァ ダプターライゲーシヨン、リン酸化、 5 '—アダプターライゲーシヨン、逆転写、 PCR増幅 、コンカテマ一化、ベクターへのライゲーシヨンを順次経て、低分子 RNAをクローニン グし、そのクローンの塩基配列を決定する方法等があげられる。あるいは、例えば、 サイエンス（Science) 294, 858-862 (2001)に記載の方法に準じて、 15%ポリアクリルァ ミドゲル電気泳動による低分子 RNAの分離及び切り出し、 5 ' アデニル化 3 '—ァダ プターライゲーシヨン、 5 ' アダプターライゲーシヨン、逆転写、 PCR増幅、コンカテマ 一化、ベクターへのライゲーシヨンを順次経て、低分子 RNAをクローユングし、そのク ローンの塩基配列を決定する方法等があげられる。  [0079] As a method for obtaining low molecular weight RNA, specifically, 15% polyacrylamide gel electrophoresis was performed according to the method described in Jeans & Development 15, 188-200 (2000). Separation and excision of small RNA by 5 'end dephosphorylation, 3' adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, concatamerization, ligation to vector The method of cloning low molecular weight RNA and determining the base sequence of the clone, etc. can be mentioned. Alternatively, for example, in accordance with the method described in Science 294, 858-862 (2001), separation and excision of small RNA by 15% polyacrylamide gel electrophoresis, 5 ′ adenylation 3′-adapter ligation Examples include a method of clotting a low molecular weight RNA and determining the base sequence of the clone through a sequence of gating, 5 ′ adapter ligation, reverse transcription, PCR amplification, concatenation, and ligation to a vector.

[0080] あるいは、 Nucleic Acids Research 34， 1765-1771 (2006)に記載の方法により、 15% ポリアクリルアミドゲル電気泳動による低分子 RNAの分離及び切り出し、 5'末端脱リン 酸化、 3' アダプターライゲーシヨン、リン酸化、 5' アダプターライゲーシヨン、逆転 写、 PCR増幅、マイクロビーズベクターへのライゲーシヨンを順次経て、低分子 RNAを クローユングし、そのマイクロビーズの塩基配列を読み取ることで塩基配列を決定す ることにより低分子 RNAを取得すること力 Sできる。 [0080] Alternatively, according to the method described in Nucleic Acids Research 34, 1765-1771 (2006), 15% Separation and excision of small RNA by polyacrylamide gel electrophoresis, 5 'terminal dephosphorylation, 3' adapter ligation, phosphorylation, 5 'adapter ligation, reverse copy, PCR amplification, ligation to microbead vector Through this process, low-molecular-weight RNA can be obtained by cloning low-molecular-weight RNA and determining the base sequence by reading the base sequence of the microbeads.

[0081] また、 small RNA Cloning Kit (タカラバイオ社製）を用いて低分子 RNAを取得するこ ともできる。 [0081] In addition, small RNA can be obtained using a small RNA Cloning Kit (manufactured by Takara Bio Inc.).

(1 3)マイクロ RNAの同定  (1 3) Identification of microRNA

取得した低分子 RNA配列がマイクロ RNAであるかは、アール ェヌ エイ（RNA)，9 ， 277-279 (2003)に記載の基準に従うか否かで判定できる。例えば、上記方法で取 得して塩基配列を決定した低分子 RNAの場合、以下のようにしておこなうことができる Whether or not the obtained small RNA sequence is a microRNA can be determined by following the criteria described in RNA (RNA), 9, 277-279 (2003). For example, in the case of a low molecular weight RNA obtained by the above method and the base sequence determined, it can be carried out as follows.

〇 Yes

[0082] すなわち、取得した低分子 RNA塩基配列に対応する DNA配列を 5'末端側および 3 '末端側にそれぞれ 50 nt程度伸ばした周辺ゲノム配列を取得し、そのゲノム配列か ら転写されることが予測される RNAの 2次構造を予測する。その結果、ヘアピン構造を 有し、且つ該低分子 RNAの塩基配列がヘアピンの片鎖に位置する場合、該低分子 R NAはマイクロ RNAであると判定できる。ゲノム配列は一般に公開されており、例えば 、 UCSC Genome Bioinformatics (http://genome.ucsc.edu/)力、ら入手 4目 である。ま た、 2次構造予測も様々なプログラムが公開されており、例えば、 RNAfold [Nucleic A cids Research 31， 3429—3431 (2003)]や Mfold [Nucleic Acids Research 31， 3406—341 5 (2003)]等を用いること力 Sできる。また、既存のマイクロ RNA配列は miRBase (11570 97436265.13)というデータベースに登録されており、ここに記載の配列と同一か否か で、既存のマイクロ RNAと同一か否かを判定することができる。  That is, a peripheral genomic sequence obtained by extending the DNA sequence corresponding to the obtained small RNA base sequence by about 50 nt to the 5 ′ end side and the 3 ′ end side, respectively, is obtained and transcribed from the genomic sequence. Predicts secondary structure of RNA. As a result, when it has a hairpin structure and the base sequence of the low molecular RNA is located on one strand of the hairpin, it can be determined that the low molecular RNA is a microRNA. Genome sequences are publicly available, for example, UCSC Genome Bioinformatics (http://genome.ucsc.edu/), etc. Various programs for secondary structure prediction are also available, such as RNAfold [Nucleic Acids Research 31, 3429-3431 (2003)] and Mfold [Nucleic Acids Research 31, 3406-341 5 (2003)]. The power S can be used. In addition, existing microRNA sequences are registered in a database called miRBase (11570 97436265.13), and whether or not they are identical to existing microRNAs can be determined based on whether or not they are identical to the sequences described here.

[0083] 上記の方法で同定した、間葉系幹細胞で発現するマイクロ RNAとして、例えば、配 列番号 1〜5のいずれかで表される塩基配列からなる核酸をあげることができる。さら に、配列番号 1〜5のいずれかで表されるヒトマイクロ RNAと同一の機能を有するマ イク口 RNAとして、該ヒトマイクロ RNAのォーソログである、配列番号 6〜65で表され る塩基配列からなる核酸をあげることができる。 [0084] また、マイクロ RNAがその標的遺伝子の mRNAの翻訳を抑制するメカニズムとして、 マイクロ RNAの 5'末端側 2〜8番目の塩基配列に対して相補的な塩基配列を有する mRNAは、マイクロ RNAの標的遺伝子として認識され、その翻訳がマイクロ RNAによ り抑制されることが知られている [Current Biology, 15, R458-R460 (2005)]。配列番 号 1〜5で表されるヒトマイクロ RNAの 5'末端側 2〜8番目と同じ塩基配列を有するマ イク口 RNAは、同じ mRNAの翻訳を抑制して同様の機能を有すると考えられる。具体 的な例として、配列番号 66〜93で表される塩基配列からなる核酸をあげることができ [0083] Examples of microRNAs that are identified by the above method and expressed in mesenchymal stem cells include nucleic acids having the base sequence represented by any one of SEQ ID NOs: 1 to 5. Furthermore, as a microphone RNA having the same function as the human microRNA represented by any of SEQ ID NOs: 1 to 5, the nucleotide sequence represented by SEQ ID NOs: 6-65, which is an ortholog of the human microRNA The nucleic acid which consists of can be mention | raise | lifted. [0084] As a mechanism by which microRNA suppresses translation of mRNA of its target gene, mRNA having a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is It is known that it is recognized as a target gene of E. coli and its translation is known to be suppressed by microRNA [Current Biology, 15, R458-R460 (2005)]. Micromouth RNA having the same base sequence as the 2nd to 8th positions on the 5 ′ end of human microRNA represented by SEQ ID NOs: 1 to 5 is considered to have the same function by suppressing translation of the same mRNA. . As a specific example, a nucleic acid having a base sequence represented by SEQ ID NOs: 66 to 93 can be mentioned.

(1 -4)マイクロ RNA前駆体の同定 (1 -4) Identification of microRNA precursors

上記（1 3)で同定したマイクロ RNAの塩基配列をもとに、マイクロ RNAを含むゲ ノム配列をマイクロ RNA前駆体として同定することができる。配列番号；!〜 5のいずれ かで表され、間葉系幹細胞で発現するマイクロ RNAの前駆体としては、例えば、配 列番号 94〜99のいずれかで表される塩基配列からなる核酸をあげることができる。 また、配列番号 6〜93で表されるマイクロ RNAの前駆体としては、例えば、配列番号 100〜201で表される塩基配列からなる核酸をあげることができる。  Based on the base sequence of microRNA identified in (13) above, a genomic sequence containing microRNA can be identified as a microRNA precursor. Examples of microRNA precursors represented by any one of SEQ ID NOs:! To 5 and expressed in mesenchymal stem cells include nucleic acids having a base sequence represented by any one of SEQ ID NOs: 94 to 99 be able to. Examples of the microRNA precursors represented by SEQ ID NOs: 6 to 93 include nucleic acids having the base sequences represented by SEQ ID NOs: 100 to 201.

2.核酸の合成  2.Nucleic acid synthesis

上記 1のようにして一旦間葉系幹細胞で発現するマイクロ RNAおよびマイクロ RN A前駆体が同定され、その塩基配列が決定された後は、塩基配列に基づいてリボヌ クレオチドの重合体である RNAだけでなぐデォキシリボヌクレオチドの重合体である DNAを合成すること力 Sできる。例えば、上記 1で同定した RNAの塩基配列をもとに、 D NAの塩基配列を決定することができる。 RNAの塩基配列に対応する DNAの塩基配 歹 IJは、 RNAの配列に含まれる U (ゥラシル)を T (チミン）に読み替えることで一義的に 決定できる。  After identifying microRNA and microRNA precursors that are once expressed in mesenchymal stem cells as described in 1 above and determining their base sequences, only RNA that is a ribonucleotide polymer based on the base sequence is identified. The ability to synthesize DNA, a polymer of deoxyribonucleotides. For example, based on the RNA base sequence identified in 1 above, the DNA base sequence can be determined. The DNA base sequence IJ corresponding to the RNA base sequence can be uniquely determined by replacing U (uracil) contained in the RNA sequence with T (thymine).

[0085] また、リボヌクレオチドとデォキシリボヌクレオチドが混合した重合体や、ヌクレオチド 類似体を含む重合体も同様にして合成することができる。  [0085] A polymer in which ribonucleotides and deoxyribonucleotides are mixed or a polymer containing nucleotide analogues can be synthesized in the same manner.

本発明に用いられる核酸を合成する方法としては、特に限定されず、公知の化学 合成を用いる方法、あるいは、酵素的転写法等にて製造することができる。公知の化 学合成を用いる方法として、ホスホロアミダイト法、ホスフォロチォエート法、ホスホトリ エステル法等をあげることができ、例えば、 ABI3900ハイスループット核酸合成機（ァ プライドバイオシステムズ社製）により合成すること力 Sできる。酵素的転写法としては、 目的の塩基配列を有したプラスミドまたは DNAを铸型として典型的なファージ RNAポ リメラーゼ、例えば、 T7、 Τ3、または SP6 RNAポリメラーゼを用いた転写をあげることが できる。 The method for synthesizing the nucleic acid used in the present invention is not particularly limited, and the nucleic acid can be produced by a method using a known chemical synthesis or an enzymatic transcription method. Known methods using chemical synthesis include the phosphoramidite method, the phosphorothioate method, and the phosphorotriate method. For example, the ability to synthesize with an ABI3900 high-throughput nucleic acid synthesizer (manufactured by Applied Biosystems) can be mentioned. Examples of the enzymatic transcription method include transcription using a typical phage RNA polymerase, for example, T7, Τ3, or SP6 RNA polymerase, with a plasmid or DNA having the desired base sequence as a cage.

3. マイクロ RNAおよびマイクロ RNA前駆体の機能を検出する方法  3. Methods for detecting the function of microRNA and microRNA precursors

マイクロ RNAは、ヘアピン構造を有するマイクロ RNA前駆体力 細胞質内で RNase III endonucleaseの一種である Dicerと呼ばれる蛋白質によるプロセシングを経て生成 され、その標的塩基配列を有する mRNAの翻訳を抑制する。従って、該機能を有して いるか否かで得られた核酸がマイクロ RNAであるか否かを検出することができる。  MicroRNA is a precursor of microRNA that has a hairpin structure. MicroRNA is produced through processing by a protein called Dicer, which is a kind of RNase III endonuclease, in the cytoplasm, and suppresses translation of mRNA having the target base sequence. Therefore, it is possible to detect whether or not the nucleic acid obtained based on whether or not it has the function is a microRNA.

[0086] 例えば、一本鎖 RNA力 SRNaselll endonucleaseによりプロセシングされるか否かで機 能を測定すること力できる。具体的には、機能を検出したい一本鎖 RNAを RNaselll en donucleaseと反応させ、反応産物を電気泳動した際、マイクロ RNA前駆体としての機 能を有していれば、プロセシングを受けた 20-25塩基長程度のバンドが検出されるこ とにより、マイクロ RNAの機能を有するかを検出することができる。 RNaselll endonucl easeは、マイクロ RNA前駆体をプロセシングする活性を有するものであれば特に限 定されないが、 Dicer蛋白質を用いることが好ましい。具体的には、 si-RNAse IIITM ( タカラバィォ社製）、 Cold Shock- DICER (タカラバイオ社製）、 Recombinant Dicer Enz yme (Stratagene社製)、 BLOCK- iT Dicer RNAi Transfection Kit (Invitrogen社 )、 X -treme GENE siRNA Dicer Kit (ロシュ.アプライド 'サイエンス社製）などより入手でき 、反応条件は添付の条件に従うことで実施できる。  [0086] For example, it is possible to measure the function based on whether single-stranded RNA force is processed by SRNaselll endonuclease. Specifically, if a single-stranded RNA whose function is to be detected is reacted with RNaselll endonuclease and the reaction product is electrophoresed, if it has a function as a microRNA precursor, it is processed 20- By detecting a band with a length of about 25 bases, it can be detected whether it has a microRNA function. RNaselll endonucl ease is not particularly limited as long as it has an activity to process a microRNA precursor, but it is preferable to use Dicer protein. Specifically, si-RNAse IIITM (Takara Bio), Cold Shock-DICER (Takara Bio), Recombinant Dicer Enzyme (Stratagene), BLOCK-iT Dicer RNAi Transfection Kit (Invitrogen), X- It can be obtained from treme GENE siRNA Dicer Kit (Roche Applied Science), etc., and the reaction conditions can be carried out according to the attached conditions.

[0087] マイクロ RNAの機能を検出する別の方法としては、マイクロ RNAの標的塩基配列 を有する mRNAの翻訳を抑制するか否かで機能を測定する方法をあげることができる [0087] As another method for detecting the function of microRNA, there can be mentioned a method for measuring the function depending on whether or not the translation of mRNA having the target base sequence of microRNA is suppressed.

〇 Yes

マイクロ RNAは、その標的塩基配列を 3 '末端側 untranslated region (3 ' UTR)に含 む mRNAの翻訳を抑制することが知られている [Current Biology, 15, R458-R460 (20 05)]。そこで、測定しょうとする一本鎖 RNAに対する標的塩基配列を、適当なレポ一 ター遺伝子発現ベクターの 3 ' UTRに揷入した DNAを作製して、発現ベクターに適合 した宿主細胞に導入し、その細胞に一本鎖 RNAを発現させた時に、レポーター遺伝 子の発現を測定することで、マイクロ RNAの機能を有して!/、るか否かを検出すること ができる。 MicroRNAs are known to suppress translation of mRNA containing the target nucleotide sequence in the 3 'terminal untranslated region (3' UTR) [Current Biology, 15, R458-R460 (20 05)]. Therefore, prepare a DNA in which the target base sequence for the single-stranded RNA to be measured is inserted into the 3 ′ UTR of an appropriate reporter gene expression vector, and conform to the expression vector. When it is introduced into a host cell and single-stranded RNA is expressed in that cell, the expression of the reporter gene is measured to detect whether it has a microRNA function! Can do.

[0088] レポーター遺伝子発現ベクターは、レポーター遺伝子の上流にプロモーターを有し ており、宿主細胞においてレポーター遺伝子が発現できるものであればいかなるもの でもよい。レポーター遺伝子としては、あらゆるレポーター遺伝子を使用することが可 能であるが、例えば、ホタル 'ルシフェラーゼ遺伝子、ゥミシィタケ 'ルシフェラーゼ遺 伝子、クロラムフエ二コール'ァセチルトランスフェラーゼ遺伝子、 /3—グルクロニダ一 ゼ遺伝子、 /3—ガラクトシダーゼ遺伝子、 /3 -ラクタマーゼ遺伝子、ェクオリン遺伝子 、グリーン 'フルォレツセント'プロテイン遺伝子および DsRed蛍光遺伝子などが利用 できる。こうした性質を有するレポーター遺伝子発現ベクターとして、例えば、 psiCHE CK-1 (Promega社製）、 psiCHECK-2 (Promega社製）、 pGL3_Control (Promega社製） 、 pGL4 (Promega社製）、 pRNAi-GL (タカラバイオ社製）、 pCMV-DsRed-Express (CL ONTECH社製）等を例示することができる。また、一本鎖 RNAは、後述の 6に記載し た方法で発現させることができる。  [0088] The reporter gene expression vector may be any vector as long as it has a promoter upstream of the reporter gene and can express the reporter gene in the host cell. As the reporter gene, any reporter gene can be used.For example, firefly luciferase gene, renilla luciferase gene, chloramphenicol acetyltransferase gene, / 3-glucuronidase gene, The / 3—galactosidase gene, the / 3-lactamase gene, the equolin gene, the green 'fluorescent' protein gene and the DsRed fluorescent gene can be used. Reporter gene expression vectors having such properties include, for example, psiCHE CK-1 (Promega), psiCHECK-2 (Promega), pGL3_Control (Promega), pGL4 (Promega), pRNAi-GL (Takara) Bio) and pCMV-DsRed-Express (CL ONTECH). Single-stranded RNA can be expressed by the method described in 6 below.

[0089] 具体的には以下のようにして検出することができる。まず宿主細胞をマルチウエル プレート等に培養し、標的配列を有したレポーター遺伝子発現ベクターと一本鎖 RN Aを発現させる。その後、レポーター活性を測定し、一本鎖 RNAを発現させない場合 に比べて、一本鎖 RNAを発現させた場合のレポーター活性を測定することで、マイク 口 RNAの機倉を検出すること力 Sできる。  Specifically, it can be detected as follows. First, host cells are cultured in a multiwell plate or the like, and a reporter gene expression vector having a target sequence and single-stranded RNA are expressed. After that, the reporter activity is measured and the ability to detect the mechanism of the microphone RNA is measured by measuring the reporter activity when the single-stranded RNA is expressed compared to when not expressing the single-stranded RNA. it can.

4.マイクロ RNAおよびマイクロ RNA前駆体の 現を検出する方法  4. Method of detecting the appearance of microRNA and microRNA precursor

以下に、間葉系幹細胞で発現するマイクロ RNAおよびその前駆体の発現を検出 する方法について説明する。  The following describes a method for detecting the expression of microRNAs and their precursors expressed in mesenchymal stem cells.

[0090] マイクロ RNAおよびその前駆体の発現量を検出する方法としては、例えば、（1)ノ ーザンハイブリダィゼーシヨン、（2)ドットブロットハイブリダィゼーシヨン、（3) in situノヽ イブリダィゼーシヨン、（4)定量的 PCR、（5)デフアレンシャル 'ノヽイブリダィゼーシヨン 、（6)マイクロアレイ、（7)リボヌクレアーゼ保護アツセィ等が挙げられる。  [0090] Examples of methods for detecting the expression levels of microRNA and its precursor include (1) Northern hybridization, (2) Dot blot hybridization, and (3) In situ detection. Examples include hybridization, (4) quantitative PCR, (5) differential differential hybridization, (6) microarray, and (7) ribonuclease protection assay.

[0091] ノーザンハイブリダィゼーシヨン法とは、検体由来 RNAをゲル電気泳動で分離後、 ナイロンフィルタ一等の支持体に転写し、該マイクロ RNAまたはマイクロ RNA前駆体 の塩基配列をもとに適宜標識をしたプローブを作製し、ノ、イブリダィゼーシヨンおよび 洗浄をおこなうことで、特異的に結合したバンドを検出する方法であり、具体的には、 例えば、 Science, 294， 853-858 (2001)に記載の方法等に従って行うことができる。 [0091] The Northern hybridization method is a method in which sample-derived RNA is separated by gel electrophoresis, Transfer to a support such as a nylon filter, prepare a probe with appropriate labeling based on the base sequence of the microRNA or microRNA precursor, and perform specific identification by washing, hybridization, and washing. This is a method for detecting a bound band, specifically, for example, according to the method described in Science, 294, 853-858 (2001).

[0092] 標識プローブは、例えば、ニック'トランスレーション、ランダム ·プライミングまたは 5' 末端のリン酸化等の方法により放射性同位体、ビォチン、ジゴキシゲニン、蛍光基、 化学発光基等を、該マイクロ RNAまたはマイクロ RNA前駆体の塩基配列と相補的 な配列を有する DNAや RNA、あるいは LNAに取り込ませることで調製できる。標識プ ローブの結合量は該マイクロ RNAまたはマイクロ RNA前駆体の発現量を反映するこ とから、結合した標識プローブの量を定量することで該マイクロ RNAまたはマイクロ R NA前駆体の発現量を定量することができる。電気泳動、メンブレンの移行、プローブ の調製、ハイブリダィゼーシヨン、核酸の検出については、モレキユラ一'クローニング 第 3版に記載の方法により行うことができる。  [0092] The labeled probe may be a radioisotope, biotin, digoxigenin, a fluorescent group, a chemiluminescent group, etc. by the method such as nick 'translation, random priming or phosphorylation at the 5' end. It can be prepared by incorporating it into DNA, RNA, or LNA having a sequence complementary to the base sequence of the RNA precursor. Since the binding amount of the labeled probe reflects the expression level of the microRNA or microRNA precursor, the expression level of the microRNA or microRNA precursor is quantified by quantifying the amount of bound labeled probe. can do. Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be carried out by the methods described in Molecular 3 ′ Cloning 3rd edition.

[0093] ドットブロットハイブリダィゼーシヨンは、組織や細胞から抽出した RNAをメンブレン 上に点状にスポットして固定し、プローブとなる標識したポリヌクレオチドとハイブリダ ィゼーシヨンを行レ、、プローブと特異的にハイブリダィズする RNAを検出する方法で ある。プローブとしてはノーザンハイブリダィゼーシヨンと同様のものを用いることがで きる。 RNAの調製、 RNAのスポット、ハイブリダィゼーシヨン、 RNAの検出については、 モレキュラー 'クローニング第 3版に記載の方法により行なうことができる。  [0093] In dot blot hybridization, RNA extracted from tissues and cells is spot-fixed on a membrane in the form of spots, and labeled polynucleotides that serve as probes are hybridized with specific probes. This is a method for detecting RNA that hybridizes in a normal manner. A probe similar to Northern hybridization can be used. RNA preparation, RNA spots, hybridization, and RNA detection can be performed by the methods described in Molecular Cloning 3rd edition.

[0094] in situハイブリダィゼーシヨンは、生体から取得した組織のパラフィンまたはクリオス タツト切片、あるいは固定化した細胞を検体として用い、標識したプローブとハイプリ ダイゼーシヨンならびに洗浄の工程を行い、顕微鏡観察により、該マイクロ RNAまた はマイクロ RNA前駆体の組織や細胞内での分布や局在を調べる方法である [Metho ds in Enzymology, 254， 419 (1995)]。プローブとしてはノーザンハイブリダィゼーショ ンと同様のものを用いることができる。具体的には、 Nature Method, 3， 27 (2006)に記 載の方法に従って、マイクロ RNAを検出すること力 Sできる。  [0094] In situ hybridization uses a paraffin or cryostat section of tissue obtained from a living body or immobilized cells as a specimen, and performs a labeled probe, a hybridization, and a washing step. This is a method for examining the distribution and localization of the microRNA or microRNA precursor in tissues and cells [Metho ds in Enzymology, 254, 419 (1995)]. A probe similar to Northern hybridization can be used. Specifically, it is possible to detect microRNA according to the method described in Nature Method, 3, 27 (2006).

[0095] 定量的 PCRでは、検体由来 RNAから、逆転写用プライマーと逆転写酵素を用いて 合成した cDNA (以後、該 cDNAを検体由来 cDNAと称する）を測定に用いる。 cDNA合 成に供する逆転写用プライマーとして、ランダムプライマーあるいは特異的 RTプライ マー等を用いることができる。特異的 RTプライマーとは、本発明に用いるマイクロ RN Aまたはマイクロ RNA前駆体、およびその周辺ゲノム配列に対応する塩基配列に相 補する配列を有するプライマーをレ、う。 [0095] In quantitative PCR, cDNA synthesized from a sample-derived RNA using a reverse transcription primer and a reverse transcriptase (hereinafter, the cDNA is referred to as a sample-derived cDNA) is used for measurement. cDNA synthesis A random primer or a specific RT primer can be used as a reverse transcription primer for the preparation. The specific RT primer refers to a primer having a sequence complementary to the base sequence corresponding to the microRNA or microRNA precursor used in the present invention and its surrounding genomic sequence.

[0096] 例えば、検体由来 cDNAを合成後、これを铸型とし、該 cDNAまたはマイクロ RNA前 駆体、およびその周辺ゲノム配列に対応する塩基配歹 lj、あるいは逆転写用プライマ 一に対応する塩基配列から設計した铸型特異的なプライマーを用いて PCRを行い、 マイクロ RNAまたはマイクロ RNA前駆体の一部または全部を含む cDNAの断片を増 幅させ、ある一定量に達するまでのサイクル数から検体由来 RNAに含まれる該マイク 口 RNAまたはマイクロ RNA前駆体を含有する核酸の量を検出する。铸型特異的な プライマーとしては、該マイクロ RNAまたはマイクロ RNA前駆体、およびその周辺ゲ ノム配列に対応する適当な領域を選択し、その領域の塩基配列の 5'端 20〜40塩基 の配列からなる DNAまたは LNA、および 3'端 20〜40塩基と相補的な配列からなる DN Aまたは LNAの糸且を用いること力 Sできる。具体的には、 Nucleic Acids Research, 32， e4 3 (2004)に記載の方法等に準じて行うことができる。  [0096] For example, after synthesizing a sample-derived cDNA, this is converted into a cage shape, and the nucleotide sequence lj corresponding to the cDNA or microRNA precursor and its surrounding genomic sequence, or the base corresponding to the reverse transcription primer. PCR is performed using a type-specific primer designed from the sequence to amplify a fragment of cDNA containing part or all of the microRNA or microRNA precursor, and the number of cycles until a certain amount is reached. The amount of nucleic acid containing the microRNA or microRNA precursor contained in the derived RNA is detected. As the type-specific primer, an appropriate region corresponding to the microRNA or microRNA precursor and its surrounding genomic sequence is selected, and the 5 ′ end 20-40 base sequence of the base sequence of the region is selected. It is possible to use DNA or LNA, and DNA or LNA consisting of a sequence complementary to 20 to 40 bases at the 3 ′ end. Specifically, it can be carried out according to the method described in Nucleic Acids Research, 32, e4 3 (2004).

[0097] または、 cDNA合成に供する逆転写用プライマーとして、ステム'ループ構造を有し た特異的 RTプライマーを用いることもできる。具体的には、 Nucleic Acid Research, 3 3， el79 (2005)に記載の方法、あるいは、 TaqMan MicroRNA Assays (アプライドバイ ォシステムズ社製）を用いて行うことができる。  [0097] Alternatively, a specific RT primer having a stem'loop structure can also be used as a reverse transcription primer for cDNA synthesis. Specifically, it can be performed using the method described in Nucleic Acid Research, 33, el79 (2005), or TaqMan MicroRNA Assays (Applied Systems).

更に、マイクロ RNAまたはマイクロ RNA前駆体の 1塩基以上の塩基配列に対応す る DNAある!/、は LNAを固定化させたフィルターある!/、はスライドガラスやシリコンなど の基盤に対して、検体由来 cDNAをノヽイブリダィゼーシヨンし、洗浄を行うことにより、 該マイクロ RNAまたはマイクロ RNA前駆体の量の変動を検出することができる。この ようなハイブリダィゼーシヨンに基づく方法には、ディファレンシャル 'ハイブリダィゼー シヨン [Trends Genet., 7， 314 (1991)]やマイクロアレイ [Genome Res., 6， 639 (1996)] を用いる方法があげられる。いずれの方法もフィルターあるいは基盤上に U6 RNAな どに対応する塩基配列を有する核酸を内部標準コントロールとして固定化することで 、対照検体と標的検体の間での本発明の核酸の量の違いを正確に検出することがで きる。また対照検体と検体由来の RNAをもとにそれぞれ異なる標識の dNTP (dATP、 d GTP、 dCTP、 dTTPの混合物）を用いて標識 cDNA合成を行い、 1枚のフィルターある いは 1枚の基盤に 2つの標識 cDNAを同時にハイブリダィズさせることで正確な該マイ クロ RNAまたはマイクロ RNA前駆体の定量を行うことができる。例えば、 Proc.Natl.A cad.Sci. USA, 101， 9740-9744 (2004)や Nucleic Acid Research, 32， el88 (2004)等に 記載のマイクロアレイを用いてマイクロ RNAを検出することができる。具体的には、 mi rVana miRNA Bioarray (Ambion社製）と同様にして検出または定量することができる。 In addition, there is DNA corresponding to the base sequence of one or more bases of microRNA or microRNA precursor! / Is a filter with LNA immobilized! /, Is a specimen against a substrate such as a glass slide or silicon. A change in the amount of the microRNA or the microRNA precursor can be detected by performing hybridization on the derived cDNA and washing. Examples of such hybridization-based methods include a method using a differential 'hybridization [Trends Genet., 7, 314 (1991)] or a microarray [Genome Res., 6, 639 (1996)]. . In either method, a nucleic acid having a nucleotide sequence corresponding to U6 RNA or the like is immobilized on a filter or substrate as an internal standard control, so that the difference in the amount of the nucleic acid of the present invention between the control sample and the target sample is eliminated. Can be detected accurately wear. Labeled cDNA is synthesized using dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) that are labeled differently based on RNA from the control sample and sample, and can be used on a single filter or single substrate. By accurately hybridizing two labeled cDNAs, the microRNA or microRNA precursor can be accurately quantified. For example, microRNA can be detected using a microarray described in Proc. Natl. Acad. Sci. USA, 101, 9740-9744 (2004), Nucleic Acid Research, 32, el88 (2004), and the like. Specifically, it can be detected or quantified in the same manner as mirVana miRNA Bioarray (Ambion).

[0098] リボヌクレアーゼ保護アツセィでは、まずマイクロ RNAまたはマイクロ RNA前駆体、 あるいはその周辺ゲノム配列に対応する塩基配列の 3'端に T7プロモーター、 SP6プロ モーターなどのプロモーター配列を結合し、標識した NTP (ATP、 GTP、 CTP、 UTPの 混合物）および RNAポリメラーゼを用いたイン'ビトロの転写系により、標識したアンチ センス RNAを合成する。該標識アンチセンス RNAを、検体由来 RNAと結合させて、 RN A-RNAノヽイブリツドを形成させた後、 1本鎖 RNAのみを分解するリボヌクレアーゼ Aで 消化する。該消化物をゲル電気泳動し、 RNA-RNAハイブリッドを形成することにより 消化から保護された RNA断片を、該マイクロ RNAまたはマイクロ RNA前駆体として、 検出または定量する。具体的には、 mirVana miRNA Detection Kit (Ambion社製）を 用いて検出または定量することができる。 [0098] In the ribonuclease protection assay, first, a promoter sequence such as a T7 promoter or SP6 promoter is bound to the 3 ′ end of the base sequence corresponding to the microRNA or microRNA precursor, or its surrounding genomic sequence, and labeled NTP ( A labeled antisense RNA is synthesized by an in vitro transcription system using a mixture of ATP, GTP, CTP and UTP) and RNA polymerase. The labeled antisense RNA is bound to RNA derived from a sample to form an RNA-RNA oligonucleotide, and then digested with ribonuclease A that degrades only single-stranded RNA. The digested product is subjected to gel electrophoresis, and an RNA fragment protected from digestion by forming an RNA-RNA hybrid is detected or quantified as the microRNA or microRNA precursor. Specifically, it can be detected or quantified using mirVana miRNA Detection Kit (Ambion).

5. マイクロ RNAおよびマイクロ RNA前駆体の栾 を掄出する方法  5. How to detect microRNA and microRNA precursors

本発明に用いるマイクロ RNAおよびマイクロ RNA前駆体の変異を検出する方法と して、正常型のマイクロ RNAと変異型のマイクロ RNA、および正常型のマイクロ RN A前駆体と変異型のマイクロ RNA前駆体とのハイブリダィズにより形成されるへテロ 二本鎖を検出する方法を用いることができる。  Methods for detecting mutations in microRNA and microRNA precursors used in the present invention include normal microRNA and mutant microRNA, and normal microRNA precursor and mutant microRNA precursor. And a method for detecting a heteroduplex formed by hybridization.

[0099] ヘテロ二本鎖を検出する方法としては、（1)ポリアクリルアミドゲル電気泳動による ヘテロ二本鎖検出法 [Trends genet., 7， 5 (1991)]、（2)—本鎖コンフオメーシヨン多 型解析法 [Genomics, 16， 325-332 (1993)]、（3)ミスマッチの化学的切断法（CCM， c hemical cleavage or mismatchesノ [numan Genetics (1996), Ί om Strachan and Andre w P. Read, BIOS Scientific Publishers Limited], (4)ミスマッチの酵素的切断法 [Nat ure Genetics, 9， 103—104 (1996)]、（5)変性ゲル電気泳動法 [Mutat. Res., 288， 103 -112 (1993)]等の方法があげられる。 [0099] Methods for detecting heteroduplex include (1) heteroduplex detection by polyacrylamide gel electrophoresis [Trends genet., 7, 5 (1991)], (2) —strand conformation Chillon polymorphism analysis [Genomics, 16, 325-332 (1993)], (3) Chemical cleavage or mismatches (CCM, num om Genetics (1996), Ί om Strachan and Andre w P Read, BIOS Scientific Publishers Limited], (4) Enzymatic cleavage method of mismatch [Nature Genetics, 9, 103-104 (1996)], (5) Denaturing gel electrophoresis [Mutat. Res., 288, 103 -112 (1993)].

[0100] ポリアクリルアミドゲル電気泳動法によるへテロ二本鎖検出法は、例えば、以下のよ うにして行う。まず、検体由来 DNAあるいは検体由来 cDNAをテンプレートに対し、マ イク口 RNAまたはマイクロ RNA前駆体に対応する DNAの塩基配列を含むゲノムの 塩基配列を基に設計したプライマーにより、 200 bpよりも小さい断片として増幅する。 ヘテロ二本鎖が形成された場合は、変異を持たないホモ二本鎖よりも移動度が遅ぐ それらは余分なバンドとして検出することができる。特製のゲル（Hydro-link, MDEな ど）を用いたほうが分離度はよい。 200 bpよりも小さい断片の検索ならば、揷入、欠失 、ほとんどの 1塩基置換を検出することができる。ヘテロ二本鎖解析は、次に述べる 一本鎖コンフオメーシヨン解析と組み合わせた 1枚のゲルで行うことが望ましい。 [0100] The heteroduplex detection method by polyacrylamide gel electrophoresis is performed, for example, as follows. First, a fragment smaller than 200 bp using a primer designed based on the base sequence of the genome containing the base sequence of the DNA corresponding to the microRNA or microRNA precursor, using the sample-derived DNA or the sample-derived cDNA as a template. Amplify as. When heteroduplexes are formed, they are slower in mobility than homoduplexes without mutations and can be detected as extra bands. The degree of separation is better when special gels (Hydro-link, MDE, etc.) are used. Searching for fragments smaller than 200 bp can detect insertions, deletions, and most single base substitutions. Heteroduplex analysis is preferably performed on a single gel combined with the single-strand conformation analysis described below.

[0101] 一本鎖コンフオメーシヨン多型解析（SSCP解析； single strand conformation polymor phism analysis)では、検体由来 DNAあるいは検体由来 cDNAをテンプレートにして、 マイクロ RNAまたはマイクロ RNA前駆体に対応する DNAの塩基配列を含むゲノムの 塩基配列に基づき設計したプライマーを用いて、 200bpよりも小さい断片として増幅し た DNAを、変性後に、未変性ポリアクリルアミドゲル中で泳動する。 DNA増幅を行う際 にプライマーを同位体あるいは蛍光色素で標識する力、、または未標識の増幅産物を 銀染色することにより、増幅した DNAをバンドとして検出すること力 Sできる。野生型の パターンとの相違を明らかにするために、コントロールの検体も同時に泳動すると、変 異を持った断片を移動度の違!/、から検出できる。  [0101] In single-strand conformation polymorphism analysis (SSCP analysis), DNA bases corresponding to microRNAs or microRNA precursors using specimen-derived DNA or specimen-derived cDNA as templates. Using a primer designed based on the genomic nucleotide sequence including the sequence, DNA amplified as a fragment smaller than 200 bp is denatured and run in a native polyacrylamide gel. The ability to label primers with isotopes or fluorescent dyes during DNA amplification, or the ability to detect amplified DNA as a band by silver-staining unlabeled amplification products. In order to clarify the difference from the wild-type pattern, when a control sample is also run at the same time, a fragment with the difference can be detected from the difference in mobility.

[0102] ミスマッチ化学的切断法（CCM法）では、検体由来 DNAあるいは検体由来 cDNAを テンプレートに、マイクロ RNAまたはマイクロ RNA前駆体に対応する DNAの塩基配 歹 IJを含むゲノムの塩基配列に基づき設計したプライマーで増幅した DNA断片を、該 マイクロ RNAまたはマイクロ RNA前駆体に同位体あるいは蛍光標識をとり込ませた 標識核酸とハイブリダィズさせ、四酸化オスミウムで処理することでミスマッチしている 場所の DNAの一方の鎖を切断させ変異を検出することができる。 CCMは最も感度の 高い検出法の 1つであり、キロベースの長さの検体にも適応できる。  [0102] The mismatch chemical cleavage method (CCM method) is designed based on the base sequence of the genome containing IJ, the base sequence of DNA corresponding to microRNA or microRNA precursor, using the sample-derived DNA or sample-derived cDNA as a template. The DNA fragment amplified with the selected primer is hybridized with the labeled nucleic acid in which the microRNA or microRNA precursor is incorporated with an isotope or fluorescent label, and treated with osmium tetroxide. Mutation can be detected by cleaving one strand. CCM is one of the most sensitive detection methods and can be applied to specimens of kilobase length.

[0103] 上記、四酸化オスミウムの代わりに T4ファージリゾルベースとエンドヌクレアーゼ VII のような細胞内でミスマッチの修復に関与する酵素と RNaseAと組み合わせることで、 酵素的にミスマッチを切断することもできる。 [0103] By combining RNaseA with an enzyme involved in mismatch repair in cells such as T4 phage resol base and endonuclease VII instead of osmium tetroxide, The mismatch can also be cleaved enzymatically.

変十生ゲノレ電気泳動法（denaturing gradient gel electrophoresis： DGGE法）では、検 体由来 DNAあるいは検体由来 cDNAをテンプレートに、マイクロ RNAまたはマイクロ R NA前駆体に対応する DNAの塩基配列を含むゲノムの塩基配列に基づき設計した プライマーで増幅した DNA断片を化学的変性剤の濃度勾配や温度勾配を有するゲ ルを用いて電気泳動する。増幅した DNA断片はゲル内を一本鎖に変性する位置ま で移動し、変性後は移動しなくなる。変異がある場合とない場合では増幅した DNAの ゲル内での移動が異なることから、変異の存在を検出できる。検出感度を上げるには それぞれのプライマーにポリ (G:C)端末を付けるとよ!/、。  In denaturing gradient gel electrophoresis (DGGE method), genomic bases that contain DNA sequences corresponding to microRNA or microRNA precursors using specimen-derived DNA or specimen-derived cDNA as a template. DNA fragments amplified with primers designed based on the sequence are electrophoresed using a gel with a chemical denaturant concentration gradient or temperature gradient. The amplified DNA fragment moves to the position where it is denatured into a single strand in the gel and does not move after denaturation. The presence of the mutation can be detected because the movement of the amplified DNA in the gel differs in the presence and absence of the mutation. To increase detection sensitivity, attach a poly (G: C) terminal to each primer! /.

[0104] また、検体由来 DNAあるいは検体由来 cDNAの塩基配列を直接的に決定し、解析 することにより、マイクロ RNAまたはマイクロ RNA前駆体の変異を検出することもでき [0104] In addition, mutations in microRNA or microRNA precursors can be detected by directly determining and analyzing the base sequence of specimen-derived DNA or specimen-derived cDNA.

6. マイクロ RNAまたはマイクロ RNA前駆体を発現させる方法 6. Methods for expressing microRNAs or microRNA precursors

本発明に用いるマイクロ RNAもしくはその誘導体、マイクロ RNA前駆体もしくはそ の誘導体、または二本鎖核酸は、細胞内に導入して該マイクロ RNAもしくはその誘 導体、マイクロ RNA前駆体もしくはその誘導体、または二本鎖核酸が転写されること により、生合成されるようなベクターを用いることにより発現させること力 Sできる。具体 的には、該マイクロ RNAもしくはその誘導体、マイクロ RNA前駆体もしくはその誘導 体、または二本鎖核酸の塩基配列あるいは、その塩基配列を含むゲノムの塩基配列 をもとに、ヘアピン部分を含む DNA断片を調製し、発現ベクター内のプロモーター下 に揷入して発現プラスミドを造成し、次に該発現プラスミドを、該発現べクタ一に適合 した宿主細胞に導入することにより該マイクロ RNAもしくはその誘導体、マイクロ RN A前駆体もしくはその誘導体、または二本鎖核酸を発現させることができる。  A microRNA or derivative thereof, a microRNA precursor or derivative thereof, or a double-stranded nucleic acid used in the present invention is introduced into a cell and the microRNA or derivative thereof, a microRNA precursor or derivative thereof, or a double-stranded nucleic acid. By transcribing the double-stranded nucleic acid, it can be expressed by using a vector that is biosynthesized. Specifically, based on the base sequence of the microRNA or derivative thereof, the microRNA precursor or derivative thereof, or a double-stranded nucleic acid, or the genomic base sequence containing the base sequence, DNA containing a hairpin portion. A fragment is prepared, inserted into a promoter in an expression vector to construct an expression plasmid, and then the expression plasmid is introduced into a host cell suitable for the expression vector to thereby produce the microRNA or a derivative thereof. , MicroRNA precursors or derivatives thereof, or double stranded nucleic acids can be expressed.

[0105] 発現ベクターとしては、宿主細胞において自立複製可能または染色体中への組込 みが可能で、本発明に用いるマイクロ RNAもしくはその誘導体、マイクロ RNA前駆 体もしくはその誘導体、または二本鎖核酸の塩基配列を含む遺伝子を転写できる位 置にプロモーターを含有しているものが用いられる。プロモーターとしては、宿主細 胞中で発現できるものであれば、いかなるものでもよぐ例えば、 RNA polymerase Il(p ol Π)系プロモーターや U6 RNAや HI RNAの転写系である RNA polymerase III(pol III) 系プロモーター等をあげることができる。 pol II系プロモーターとしては例えば、サイト メガロウィルス（ヒト CMV)の IE(immediate early)遺伝子のプロモーター、 SV40の初期 プロモーター等をあげることができる。それらを用いた発現ベクターとして、例えば、 p CDNA6.2-GW/miR (Invitrogen社製）、 pSilencer 4.1_CMV(Ambion社製）等を例示 すること力 Sできる。 pol III系プロモーターとしては U6 RNAや HI RNAあるいは tRNAの プロモーターをあげることができる。それらを用いた発現ベクターとして、例えば、 pSI Nsi-hHl DNA (タカラバイオ社製）、 pSINsi_hU6 DNA (タカラバイオ社製）、 pENTR/U 6 (Invitrogen社製）等をあげることができる。 [0105] The expression vector can replicate autonomously in a host cell or can be integrated into a chromosome, and can be used for microRNA or a derivative thereof, a microRNA precursor or a derivative thereof, or a double-stranded nucleic acid used in the present invention. Those containing a promoter at a position where a gene containing the base sequence can be transcribed are used. Any promoter can be used as long as it can be expressed in the host cell.For example, RNA polymerase Il (p ol iii) type promoters and RNA polymerase III (pol III) type promoters that are transcription systems of U6 RNA and HI RNA. Examples of the pol II promoter include a promoter of cytomegalovirus (human CMV) IE (immediate early) gene, SV40 early promoter, and the like. Examples of expression vectors using them include pCDNA6.2-GW / miR (Invitrogen) and pSilencer 4.1_CMV (Ambion). Examples of pol III promoters include U6 RNA, HI RNA, and tRNA promoters. Examples of expression vectors using them include pSI Nsi-hHl DNA (Takara Bio), pSINsi_hU6 DNA (Takara Bio), pENTR / U 6 (Invitrogen) and the like.

[0106] 本発明に用いるマイクロ RNAもしくはその誘導体、マイクロ RNA前駆体もしくはそ の誘導体、または二本鎖核酸の塩基配列を含む遺伝子をウィルスベクター内のプロ モーター下流に揷入して組換えウィルスベクターを造成し、該ベクターをパッケージ ング細胞に導入して組換えウィルスを生産して、本発明に用いるマイクロ RNAもしく はその誘導体、マイクロ RNA前駆体もしくはその誘導体、または二本鎖核酸の塩基 配列を含む遺伝子を発現させることもできる。 [0106] A recombinant viral vector obtained by inserting a microRNA or a derivative thereof used in the present invention, a microRNA precursor or a derivative thereof, or a gene containing a base sequence of a double-stranded nucleic acid downstream of a promoter in a viral vector. And the vector is introduced into a packaging cell to produce a recombinant virus, and the base sequence of the microRNA or derivative thereof, microRNA precursor or derivative thereof, or double-stranded nucleic acid used in the present invention A gene containing can also be expressed.

[0107] ノ /ケージング細胞はウィルスのパッケジ一ングに必要な蛋白質をコードする遺伝 子のいずれかを欠損している組換えウィルスベクターの該欠損する蛋白質を補給で きる細胞であればいずれの細胞もよぐ例えばヒト腎臓由来の HEK293細胞、マウス繊 維芽細胞 MH3T3などを用いることができる。パッケージング細胞で補給する蛋白質と しては、レトロウイルスベクターの場合はマウスレトロウイルス由来の gag, pol, envなど の蛋白質が、レンチウィルスベクターの場合は HIVウィルス由来の gag, pol, env, vpr, vpu, vif, tat, rev, neぬどの蛋白質、アデノウイルスベクターの場合はアデノウイルス 由来の ΕΙΑ,ΕΙΒなどの蛋白質、また、アデノ随伴ウィルスベクターの場合は R印 (ρ5， ρ 19， p40), Vp(Cap)などの蛋白質を用いることができる。  [0107] A cell / caging cell may be any cell that can replenish the deficient protein of a recombinant viral vector deficient in any of the genes encoding the proteins required for viral packaging. For example, HEK293 cells derived from human kidney, mouse fibroblasts MH3T3, etc. can be used. Proteins supplemented by packaging cells include mouse retrovirus-derived gag, pol, env, etc. for retroviral vectors, and HIV virus-derived gag, pol, env, vpr for lentiviral vectors. , vpu, vif, tat, rev, ne Nude protein, in the case of adenovirus vectors, proteins such as ア デ ノ, ΕΙΒ derived from adenovirus, and R in the case of adeno-associated virus vectors (ρ5, ρ 19, p40) , Proteins such as Vp (Cap) can be used.

[0108] 発現ベクターを用いる以外にも、本発明に用いるマイクロ RNAもしくはその誘導体 、マイクロ RNA前駆体もしくはその誘導体、または二本鎖核酸を、ベクターを用いず に直接細胞に導入することもできる。本手法に用いる核酸は DNAや RNA、あるいはヌ クレオチド類似体の他、これらのキメラ分子、あるいは該核酸の誘導体も用いることが できる。具体的には、 Pre- miRTM miRNA Precursor Molecules (Ambion社製）や miRI DIAN microRNA Mimics (GEヘルスケア社製）を用いて本発明に用いるマイクロ RNA もしくはその誘導体、マイクロ RNA前駆体もしくはその誘導体、または二本鎖核酸を 発現させること力 Sでさる。 [0108] In addition to using an expression vector, the microRNA or derivative thereof, microRNA precursor or derivative thereof, or double-stranded nucleic acid used in the present invention can also be directly introduced into a cell without using a vector. In addition to DNA, RNA, or nucleotide analogues, these chimeric molecules or derivatives of these nucleic acids may be used as nucleic acids for this method. it can. Specifically, using Pre-miRTM miRNA Precursor Molecules (Ambion) or miRI DIAN microRNA Mimics (GE Healthcare), the microRNA or derivative thereof, the microRNA precursor or derivative thereof used in the present invention, or The ability to express double-stranded nucleic acids is controlled by S.

[0109] マイクロ RNAを発現させる場合は、細胞内で最終的にマイクロ RNAができる状態 になればいかなる方法でもよいが、好ましくは、（1)マイクロ RNA前駆体である一本 鎖 RNA、（2)マイクロ RNA、およびマイクロ RNAの相補鎖からなる 2本鎖 RNA、また は、（3)マイクロ RNAが Dicerに切断された後の状態を想定した 2本鎖 RNA、を導入 させる方法があげられる。具体的には、 miCENTURY OX Precursor (B-Bridge社製） 、 miCENTURY OX siMature (B-Bridge社製）、 miCENTURY OX miNatural (B-Bridge 社製）と同様にして本発明で用いるマイクロ RNAを発現させることができる。  [0109] When microRNA is expressed, any method may be used as long as microRNA can finally be produced in the cell, but preferably (1) single-stranded RNA that is a microRNA precursor, (2 Examples include a method of introducing a double-stranded RNA consisting of a microRNA and a complementary strand of microRNA, or (3) a double-stranded RNA that assumes a state after the microRNA is cut into Dicers. Specifically, the microRNA used in the present invention is expressed in the same manner as miCENTURY OX Precursor (B-Bridge), miCENTURY OX siMature (B-Bridge), and miCENTURY OX miNatural (B-Bridge). be able to.

7.マイクロ RNAおよびマイクロ RNA前駆体の発現を抑制する方法  7. Methods for suppressing the expression of microRNA and microRNA precursors

本発明に用いるマイクロ RNAまたはマイクロ RNA前駆体は、アンチセンス技術 [バ ィォサイエンスとインダストリ一， 50， 322 (1992)、化学， 46， 681 (1991)、 Biotechnology ， 9， 358 (1992)、 Trends in Biotechnology, 10， 87 (1992)、 Trends in Biotechnology, 10， 152 (1992)、細胞工学， 1 6， 1463 (1997)]、トリプル.ヘリックス技術 [Trends in Biot echnology, 10， 132 (1992)]、リホケ ム技術 [Current Opinion in Chemical Biology, 3， 274 (1999)、 FEMS Microbiology Reviews, 23， 257 (1999)、 Frontiers in Bioscience, 4， D497 (1999)、 Chemistry & Biology, 6， R33 (1999)、 Nucleic Acids Research, 26， 5 237 (1998)、 Trends In Biotechnology, 16， 438 (1998)]、デコイ DNA法 [Nippon Rinsho - Japanese Journal of Clinical Medicine, 56， 563 (1998)、 Circulation Research, 82, 1023 (1998)、 Experimental Nephrology, 5， 429 (1997)、 Nippon Rinsho - Japanese Jo urnal of Clinical Medicine, 54， 2583 (1996)]、あるいは siRNA (short interfering RNA) を用いて、その発現を抑制することができる。  The microRNA or microRNA precursor used in the present invention is an antisense technology [Bioscience and Industry, 50, 322 (1992), Chemistry, 46, 681 (1991), Biotechnology, 9, 358 (1992). , Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992), Cell Engineering, 16, 6463 (1997)], Triple Helix Technology [Trends in Biot echnology, 10, 132 (1992) )], Rehoc technology [Current Opinion in Chemical Biology, 3, 274 (1999), FEMS Microbiology Reviews, 23, 257 (1999), Frontiers in Bioscience, 4, D497 (1999), Chemistry & Biology, 6, R33 ( 1999), Nucleic Acids Research, 26, 5 237 (1998), Trends In Biotechnology, 16, 438 (1998)], Decoy DNA method [Nippon Rinsho-Japanese Journal of Clinical Medicine, 56, 563 (1998), Circulation Research, 82, 1023 (1998), Experimental Nephrology, 5, 429 (1997), Nippon Rinsho-Japanese Journal of Clinical Medicine, 54, 2583 (1996) )], Or siRNA (short interfering RNA) can be used to suppress its expression.

[0110] アンチセンスとは、ある標的核酸の塩基配列に相補的な塩基配列を有する核酸を 塩基配列特異的にハイブリダィゼーシヨンさせ、該標的核酸の発現を抑制できるもの をいう。アンチセンスに用いる核酸は DNAや RNAまたはヌクレオチド類似体の他、こ れらのキメラ分子、あるいは該核酸の誘導体も用いることができる。具体的には、 Natu re, 432， 226 (2004)等に記載の方法に従うことでアンチセンスを作製し、発現を抑制 すること力できる。具体的には、 Anti-miRTM miRNA Inhibitors (Amb ion社製）や miRI DIAN microRNA Inhibitors (GEヘルスケア社製）を用いて本発明に用いるマイクロ R NAまたはマイクロ RNA前駆体の発現を抑制することができる。 [0110] Antisense refers to a nucleic acid having a base sequence complementary to a base sequence of a certain target nucleic acid that can specifically hybridize to the base sequence to suppress the expression of the target nucleic acid. In addition to DNA, RNA, or nucleotide analogs, these chimeric molecules or derivatives of the nucleic acids can also be used as nucleic acids for antisense. Specifically, Natu By following the method described in re, 432, 226 (2004), etc., it is possible to produce an antisense and suppress the expression. Specifically, anti-miRTM miRNA Inhibitors (manufactured by Amb ion) or miRI DIAN microRNA Inhibitors (manufactured by GE Healthcare) can be used to suppress the expression of microRNA or microRNA precursor used in the present invention. it can.

[0111] siRNAとは、ある標的核酸の塩基配列を含む短い二本鎖 RNAであり、 RNA干渉（RN Ai)により、該標的核酸の発現を抑制できるものをいう。 siRNAの酉己歹 IJは、標的とする 塩基配列から文献 [Genes Dev., 13， 3191 (1999)]の条件に基づいて適宜設計するこ とができる。選択した 19塩基の配列および相補的な配列それぞれの 3'端に TTを付加 した配列を有する 2本の RNAを核酸合成機により合成し、アニーリングすることにより si RNAを作製できる。また、 pSilencer 1.0-U6 (Ambion社製）、 pSUPER (01igoEngine社 )等の siRNA発現用ベクターに上記の選択した 19塩基の配列に相当する DNAを揷入 することにより、該遺伝子の発現を抑制できる siRNAを発現するベクターを作製するこ と力 Sできる。 [0111] The siRNA is a short double-stranded RNA containing a base sequence of a certain target nucleic acid, and can suppress the expression of the target nucleic acid by RNA interference (RN Ai). The siRNA IJ can be appropriately designed based on the conditions of the literature [Genes Dev., 13, 3191 (1999)] based on the target nucleotide sequence. A siRNA can be prepared by synthesizing and annealing two RNAs each having a 19-base sequence selected and a complementary sequence each having a TT added at the 3 'end, and annealing. In addition, by inserting a DNA corresponding to the selected 19-base sequence into a siRNA expression vector such as pSilencer 1.0-U6 (Ambion) or pSUPER (01igoEngine), the expression of the gene can be suppressed. It is possible to create vectors that express siRNA.

[0112] 例えば、間葉系幹細胞で発現するマイクロ RNAまたは該マイクロ RNA前駆体に特 異的なアンチセンスまたは siRNAを用いて、間葉系幹細胞で発現するマイクロ RNA または該マイクロ RNA前駆体の発現の抑制を行うことができる。すなわち、該マイクロ RNAまたは該マイクロ RNA前駆体に特異的なアンチセンス DNAまたは siRNAを投 与することにより該マイクロ RNAの発現を抑制し、間葉系幹細胞におけるマイクロ RN Aまたはマイクロ RNA前駆体の作用を制御することができる。  [0112] For example, expression of microRNA expressed in mesenchymal stem cells or the microRNA precursor using microRNA expressed in mesenchymal stem cells or antisense or siRNA specific to the microRNA precursor Can be suppressed. That is, by applying antisense DNA or siRNA specific to the microRNA or the microRNA precursor, the expression of the microRNA is suppressed, and the action of microRNA or microRNA precursor in mesenchymal stem cells Can be controlled.

[0113] また、間葉系幹細胞で発現するマイクロ RNAまたはその前駆体の発現異常による 患者の場合、該マイクロ RNAまたはその前駆体に特異的なアンチセンスまたは siRN Aを患者に投与することにより、間葉系幹細胞の機能を制御し、上記発現異常により 発症する疾患の治療をすることができる。すなわち、該マイクロ RNAまたはその前駆 体に特異的なアンチセンスまたは siRNAは、間葉系幹細胞の異常に起因する疾患の 治療剤として有用である。  [0113] Further, in the case of a patient due to abnormal expression of microRNA or its precursor expressed in mesenchymal stem cells, by administering antisense or siRNA specific to the microRNA or its precursor to the patient, By controlling the function of mesenchymal stem cells, it is possible to treat diseases caused by the abnormal expression. That is, antisense or siRNA specific to the microRNA or its precursor is useful as a therapeutic agent for diseases caused by abnormal mesenchymal stem cells.

[0114] 該マイクロ RNAまたはその前駆体に特異的なアンチセンスまたは siRNAを上記治 療剤として使用する場合は、アンチセンスまたは siRNAを単独、あるいはレトロウィル スベクター、アデノウイルスベクター、アデノ随伴ウィルスベクターなどの適当なベクタ 一に挿入した後、下記 10、 11に記載した常法に従って医薬製剤とし、投与すること ができる。 [0114] When antisense or siRNA specific to the microRNA or its precursor is used as the above therapeutic agent, antisense or siRNA alone, or a retrovirus vector, adenovirus vector, adeno-associated virus vector, etc. Suitable vector After insertion, it can be administered as a pharmaceutical preparation according to conventional methods described in 10 and 11 below.

8. マイクロ RNAまたはマイクロ RNA前駆体の標的遺伝子の発現を抑制する方法 本発明に用いるマイクロ RNAまたはマイクロ RNA前駆体の標的遺伝子の発現を 抑制する方法としては、マイクロ RNAがその標的遺伝子の mRNAの翻訳を抑制する 活性を利用して、標的塩基配列を有した遺伝子の発現を抑制する方法であれば、い かなる方法でもよぐ例えば、マイクロ RNAの細胞内の量を増加させることにより、そ の標的遺伝子の mRNAの翻訳を抑制し、該標的遺伝子の発現を抑制する方法をあ げること力 Sできる。なお、該マイクロ RNAまたはマイクロ RNA前駆体を細胞内で発現 させるには、上記 6で記載した方法により行なうことができる。配列番号 1〜93のいず れかで表される塩基配列からなるマイクロ RNAの標的遺伝子としては、例えば、それ ぞれ前述の表 1で示される遺伝子群を例示することができる。  8. Method for suppressing expression of target gene of microRNA or microRNA precursor As a method for suppressing the expression of target gene of microRNA or microRNA precursor used in the present invention, microRNA is the mRNA of the target gene. Any method that uses the activity of suppressing translation to suppress the expression of a gene having a target base sequence can be used. For example, by increasing the intracellular level of microRNA, The ability to suppress the translation of the target gene mRNA and to suppress the expression of the target gene can be improved. The microRNA or microRNA precursor can be expressed in cells by the method described in 6 above. Examples of the target gene of the microRNA consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 93 include the gene groups shown in Table 1 above, for example.

[0115] また、表 1で示される標的遺伝子に対する siRNAを用いて、該標的遺伝子の機能を 才卬制すること力 Sでさる。 [0115] Further, using the siRNA for the target gene shown in Table 1, the ability S can control the function of the target gene.

9.間！^餘細 の增歹直および/または分化の 制剤または促進吝 II»スクリーユング すろ方法  9. Between! ^ Increase and / or enhance differentiation and / or promotion of differentiation II »Screening

本発明の間葉系幹細胞の増殖および/または分化抑制剤のスクリーニング方法とし ては、（A)本発明に用いるマイクロ RNAまたはマイクロ RNA前駆体の発現を促進さ せること、 (B)本発明に用いるマイクロ RNAの標的遺伝子の発現を抑制することを、 指標にして行う方法をあげることができる。また、本発明の間葉系幹細胞の増殖およ び/または分化促進剤のスクリーニング方法としては、 (C)本発明に用いるマイクロ R NAまたはマイクロ RNA前駆体の発現を抑制すること、 (D)本発明に用いるマイクロ RNAの標的遺伝子の発現を促進することを、指標にして行う方法をあげることができ  The method of screening for an agent for inhibiting the proliferation and / or differentiation of mesenchymal stem cells of the present invention includes (A) promoting the expression of microRNA or microRNA precursor used in the present invention, and (B) presenting the present invention. Inhibiting the expression of the target gene of the microRNA to be used can be used as an indicator. Further, the screening method for the mesenchymal stem cell proliferation and / or differentiation promoting agent of the present invention includes (C) suppressing the expression of microRNA or microRNA precursor used in the present invention, (D) Examples of the method that can be used to promote the expression of the target gene of the microRNA used in the present invention can be mentioned.

[0116] 例えば、本発明に用いられるマイクロ RNAおよびマイクロ RNA前駆体の塩基配列 から、スクリーニングの標的とする塩基配列を選択して、該塩基配列を有する核酸を 発現する細胞を利用して、選択したマイクロ RNAまたはマイクロ RNA前駆体の発現 を促進または抑制させる物質をスクリーニングすることができる。 スクリーニングに用いる、マイクロ RNAおよびマイクロ RNA前駆体の塩基配列を有 する核酸を発現する細胞としては、間葉系幹細胞のほか、上記 6で記載したように、 該塩基配列を有する核酸を発現するベクターを動物細胞や酵母などの宿主細胞に 導入して得られる形質転換細胞や、ベクターを用いずに該塩基配列を有する核酸を 直接導入した細胞等を用いることもできる。 [0116] For example, from the base sequences of the microRNA and microRNA precursor used in the present invention, a base sequence to be screened is selected and selected using a cell that expresses a nucleic acid having the base sequence. A substance that promotes or suppresses the expression of the microRNA or the microRNA precursor can be screened. The cells expressing nucleic acids having the base sequences of microRNA and microRNA precursor used for screening include mesenchymal stem cells as well as vectors expressing nucleic acids having the base sequences as described in 6 above. It is also possible to use a transformed cell obtained by introducing the DNA into a host cell such as an animal cell or yeast, or a cell into which a nucleic acid having the base sequence is directly introduced without using a vector.

[0117] 具体的なスクリーニング方法としては、（a)スクリーニングの標的とするマイクロ RNA またはマイクロ RNA前駆体の発現量の変化を指標にする方法、（b)スクリーニングの 標的とするマイクロ RNAの標的遺伝子の mRNAの発現量の変化を指標にする方法 などがあげられる。 [0117] Specific screening methods include: (a) a method that uses changes in the expression level of microRNAs or microRNA precursors targeted for screening as an index, and (b) a target gene for microRNAs that is targeted for screening. A method using as an index the change in mRNA expression level.

(a)スクリーニングの標的とするマイクロ RNAまたはマイクロ RNA前駆体の発現量の 変化を指標にするスクリーニング方法  (a) Screening method using changes in the expression level of the target microRNA or microRNA precursor as an index

該塩基配列を有する核酸を発現する細胞に対し、試験物質を接触させ、選択した 核酸の発現量の変化を指標に、間葉系幹細胞の増殖および/または分化の抑制ま たは促進させる物質を得る。核酸の発現量は、上記 3で記載した方法により検出する こと力 Sでさる。  A test substance is brought into contact with a cell that expresses a nucleic acid having the nucleotide sequence, and a substance that suppresses or promotes the proliferation and / or differentiation of mesenchymal stem cells is used as an indicator of the change in the expression level of the selected nucleic acid. obtain. The amount of nucleic acid expressed can be detected by the method S described in 3 above.

(b)スクリーニングの標的とするマイクロ RNAの標的遺伝子の mRNAの発現量の変化 を指標にするスクリーニング方法  (b) Screening method using as an index the change in the expression level of the mRNA of the target gene of the microRNA targeted for screening

標的塩基配列を有する核酸を発現する細胞に対し、試験物質を接触させ、選択し た核酸の標的遺伝子の mRNA発現量の変化を指標に、間葉系幹細胞の増殖および /または分化を抑制させる物質を得る。または、本発明に用いるマイクロ RNAに対す る標的配列を、適当なレポーター遺伝子発現ベクターの 3' UTRに揷入した DNAを作 製して、発現ベクターに適合した宿主細胞に導入し、その細胞に試験物質を接触さ せ、レポーター遺伝子の発現量の変化を指標に、間葉系幹細胞の増殖および/また は分化を抑制させる物質を得る。  A substance that suppresses the proliferation and / or differentiation of mesenchymal stem cells using a test substance in contact with a cell that expresses a nucleic acid having a target base sequence, and changes in the mRNA expression level of the target gene of the selected nucleic acid as an indicator Get. Alternatively, DNA in which the target sequence for the microRNA used in the present invention is inserted into the 3 ′ UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector. A test substance is contacted, and a substance that suppresses proliferation and / or differentiation of mesenchymal stem cells is obtained using the change in the expression level of the reporter gene as an index.

[0118] 標的塩基配列の選択は、上記 8で記載した方法により行なうことができ、配列番号 1 〜93のいずれかで表される塩基配列からなるマイクロ RNAの標的遺伝子としては、 例えば、それぞれ前述の表 1で示される遺伝子群を例示することができる。 [0118] The selection of the target base sequence can be performed by the method described in the above 8. The target gene of the microRNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 93 is, for example, as described above. The gene group shown in Table 1 can be exemplified.

10.本発明に用いられる核酸を含有する診断薬および'冶療薬 本発明に用いられる核酸、または配列番号 1〜201からなる塩基配列と相補的な 塩基配列を含む核酸は、該マイクロ RNAまたはマイクロ RNA前駆体、該マイクロ RN Aの標的遺伝子の発現を制御することにより、間葉系幹細胞等の増殖または分化の 異常等に起因する疾患の治療薬として利用することができる。また、該マイクロ RNA またはマイクロ RNA前駆体の発現、または該マイクロ RNAまたはマイクロ RNA前駆 体の変異、あるレ、は該マイクロ RNAまたはマイクロ RNA前駆体をコードするゲノムの 変異を検出することにより、間葉系幹細胞等の増殖または分化の異常等に起因する 疾患の診断薬として利用することができる。さらに、該マイクロ RNAの標的遺伝子に 対する siRNAは、該マイクロ RNAの標的遺伝子の mRNA発現を制御することにより、 間葉系幹細胞等の増殖および/または分化の異常等に起因する疾患の治療薬として 禾 IJ用すること力できる。間葉系幹細胞等の増殖または分化の異常等に起因する疾患 として、癌、骨形成不全症、軟骨形成不全症、糖尿病等をあげることができる。 10. Nucleic acid-containing diagnostics and therapeutics used in the present invention The nucleic acid used in the present invention or a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201 controls the expression of the target gene of the microRNA or microRNA precursor or the microRNA Thus, it can be used as a therapeutic agent for diseases caused by abnormal proliferation or differentiation of mesenchymal stem cells. In addition, by detecting the expression of the microRNA or microRNA precursor, or the mutation of the microRNA or microRNA precursor, or a mutation in the genome encoding the microRNA or microRNA precursor, It can be used as a diagnostic agent for diseases caused by abnormal proliferation or differentiation of leaf stem cells. Furthermore, siRNA against the target gene of the microRNA can be used as a therapeutic agent for diseases caused by abnormalities in proliferation and / or differentiation of mesenchymal stem cells by controlling the mRNA expression of the target gene of the microRNA.力 I can use IJ. Examples of the diseases caused by abnormal proliferation or differentiation of mesenchymal stem cells and the like include cancer, osteogenesis imperfecta, cartilage imperfecta and diabetes.

[0119] 本発明に用いられる核酸、または配列番号 1〜201からなる塩基配列と相補的な 塩基配列を含む核酸を有効成分として含有する診断薬は、 目的の診断法に応じて、 該マイクロ RNAまたはマイクロ RNA前駆体の定量あるいは変異の検出を行うために 必要な試薬、例えば緩衝剤、塩、反応用酵素、本発明に用いられる核酸、または配 列番号;!〜 201からなる塩基配列と相補的な塩基配列を含む核酸と結合する標識さ れた蛋白、および検出用発色剤等を含んでもよい。  [0119] A diagnostic agent containing, as an active ingredient, the nucleic acid used in the present invention or a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, the microRNA according to the target diagnostic method Or reagents necessary for quantification of microRNA precursors or detection of mutations, such as buffers, salts, reaction enzymes, nucleic acids used in the present invention, or complementary to the nucleotide sequence consisting of SEQ ID NOs:! -201 It may contain a labeled protein that binds to a nucleic acid containing a typical base sequence, a color former for detection, and the like.

[0120] 本発明に用いられる核酸、配列番号 1〜201からなる塩基配列と相補的な塩基配 列を含む核酸、または、該マイクロ RNAの標的遺伝子に対する siRNAを有効成分と して含有する治療剤は、単独で投与することもできるが、通常は薬理学的に許容され る 1つあるいはそれ以上の担体と一緒に混合し、製剤学の技術分野においてよく知ら れる任意の方法により製造した医薬製剤として投与するのが望ましい。  [0120] A therapeutic agent containing, as an active ingredient, a nucleic acid used in the present invention, a nucleic acid containing a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or siRNA against the target gene of the microRNA Can be administered alone, but is usually mixed with one or more pharmacologically acceptable carriers and prepared by any method well known in the pharmaceutical arts. It is desirable to administer as

[0121] 投与経路は、治療に際し最も効果的なものを使用するのが望ましぐ経口投与、ま たは口腔内、気道内、直腸内、皮下、筋肉内および静脈内などの非経口投与をあげ ること力 Sでき、望ましくは静脈内投与をあげることができる。  [0121] The route of administration is preferably oral, for which it is desirable to use the most effective treatment, or parenteral, such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous. It is possible to increase the dosage, preferably intravenous administration.

投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、座剤、 注射剤、軟膏、テープ剤などがあげられる。 [0122] 経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒 剤などがあげられる。 Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like. [0122] Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.

乳剤およびシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖など の糖類、ポリエチレングリコール、プロピレングリコールなどのグリコール類、ごま油、 ォリーブ油、大豆油などの油類、 P-ヒドロキシ安息香酸エステル類などの防腐剤、スト 口べリーフレーバー、ペパーミントなどのフレーバー類などを添加剤として用いて製造 できる。  Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P-hydroxybenzoate Preservatives such as acid esters, flavors such as stove leaf flavor and peppermint can be used as additives.

[0123] カプセル剤、錠剤、散剤、顆粒剤などは、乳糖、ブドウ糖、ショ糖、マンニトールなど の賦形剤、デンプン、アルギン酸ナトリウムなどの崩壊剤、ステアリン酸マグネシウム、 タルクなどの滑沢剤、ポリビュルアルコール、ヒドロキシプロピルセルロース、ゼラチン などの結合剤、脂肪酸エステルなどの界面活性剤、グリセリンなどの可塑剤などを添 加剤として用いて製造できる。  [0123] Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, It can be produced by using a binder such as bull alcohol, hydroxypropyl cellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.

[0124] 非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤などがあげられる。  [0124] Suitable formulations for parenteral administration include injections, suppositories, sprays and the like.

注射剤は、塩溶液、ブドウ糖溶液あるいは両者の混合物からなる担体などを用いて 調製される。座剤はカカオ脂、水素化脂肪またはカルボン酸などの担体を用いて調 製される。また、噴霧剤は受容者の口腔および気道粘膜を刺激せず、かつ有効成分 を微細な粒子として分散させ吸収を容易にさせる担体などを用いて調製される。  An injection is prepared using a carrier comprising a salt solution, a glucose solution or a mixture of both. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa and that facilitates absorption by dispersing the active ingredient as fine particles.

[0125] 担体として具体的には乳糖、グリセリンなどが例示される。さらには用いる担体の性 質により、エアロゾル、ドライパウダーなどの製剤が可能である。また、これらの非経口 剤においても経口剤で添加剤として例示した成分を添加することもできる。  [0125] Specific examples of the carrier include lactose and glycerin. Furthermore, preparations such as aerosols and dry powders are possible depending on the nature of the carrier used. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

投与量または投与回数は、 目的とする治療効果、投与方法、治療期間、年齢、体 重などにより異なる力 通常成人 1日当たり 10 H g/kg〜20 mg/kgである。  The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc. The usual adult dose is 10 Hg / kg to 20 mg / kg per day.

[0126] また、本発明に用いられる核酸、配列番号 1〜201からなる塩基配列と相補的な塩 基配列を含む核酸、または、マイクロ RNAの標的遺伝子に対する siRNAを有効成分 として含有する治療薬は、本発明に用いられる核酸、配列番号 1〜201からなる塩基 配列と相補的な塩基配列を含む核酸、または、マイクロ RNAの標的遺伝子に対する siRNAを発現するベクターと核酸治療薬に用いる基剤とを調合することにより製造す ることもできる [Nature Genet., 8， 42(1994)]。 [0127] 核酸治療剤に用いる基剤としては、通常注射剤に用いる基剤であればどのようなも のでもよく、蒸留水、塩化ナトリウム又は塩化ナトリウムと無機塩との混合物等の塩溶 液、マンニトール、ラタトース、デキストラン、グルコース等の溶液、グリシン、アルギニ ン等のアミノ酸溶液、有機酸溶液又は塩溶液とグルコース溶液との混合溶液等があ げられる。また常法に従い、これらの基剤に浸透圧調整剤、 pH調整剤、ゴマ油、ダイ ズ油等の植物油又はレシチンもしくは非イオン界面活性剤等の界面活性剤等の助 剤を用いて、溶液、懸濁液、分散液として注射剤を調製してもよい。これらの注射剤 を、粉末化、凍結乾燥等の操作により用時溶解用製剤として調製することもできる。 本発明の治療剤は、治療の直前に液体の場合はそのままで、個体の場合は必要に より滅菌処理をした上記の基剤に溶解して治療に使用することができる。 [0126] Further, a therapeutic agent containing, as an active ingredient, a nucleic acid used in the present invention, a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or siRNA against a target gene of microRNA A nucleic acid used in the present invention, a nucleic acid containing a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or a vector expressing siRNA against a target gene of microRNA and a base used for a nucleic acid therapeutic agent It can also be produced by blending [Nature Genet., 8, 42 (1994)]. [0127] The base used in the nucleic acid therapeutic agent may be any base that is usually used for injections, and is a salt solution such as distilled water, sodium chloride, or a mixture of sodium chloride and an inorganic salt. , Mannitol, ratatoose, dextran, glucose and the like, amino acid solutions such as glycine and arginine, organic acid solutions or mixed solutions of a salt solution and a glucose solution, and the like. In accordance with a conventional method, these bases are mixed with an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil and die oil, or an auxiliary agent such as a lecithin or a surfactant such as a nonionic surfactant. An injection may be prepared as a suspension or dispersion. These injections can also be prepared as preparations for dissolution upon use by operations such as pulverization and freeze-drying. The therapeutic agent of the present invention can be used for treatment as it is in the case of a liquid just before the treatment, and in the case of an individual, it can be dissolved in the above sterilized base as necessary.

[0128] 本発明に用いられる核酸、配列番号 1〜201からなる塩基配列と相補的な塩基配 列を含む核酸、または、マイクロ RNAの標的遺伝子に対する siRNAを含有するべクタ 一は、上記 6で作製した組換えウィルスベクターウィルスベクターをあげることができ、 より具体的には、レトロウイルスベクター及びレンチウィルスベクター等をあげることが できる。  [0128] The nucleic acid used in the present invention, a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence consisting of SEQ ID NOs: 1 to 201, or a vector containing siRNA against a target gene of microRNA is the above 6 Examples of the prepared recombinant virus vector include virus vectors, and more specifically, a retrovirus vector and a lentivirus vector.

[0129] 例えば、本発明に用いられる核酸、配列番号 1〜201からなる塩基配列と相補的な 塩基配列を含む核酸、または、マイクロ RNAの標的遺伝子に対する siRNAを、アデノ ウィルス .へキソン蛋白質に特異的なポリリジン—コンジュゲート抗体と組み合わせて コンプレックスを作製し、得られたコンプレックスをアデノウイルスベクターに結合させ ることにより、ウィルスベクターを調製することができる。該ウィルスベクターは安定に 目的の細胞に到達し、エンドソームによる細胞内に取り込まれ、細胞内で分解され核 酸を効率的に発現させることができる。  [0129] For example, a nucleic acid used in the present invention, a nucleic acid containing a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or a siRNA against a microRNA target gene is specific to an adenovirus .hexon protein A viral vector can be prepared by preparing a complex by combining with a typical polylysine-conjugated antibody and binding the resulting complex to an adenoviral vector. The viral vector stably reaches the target cell, is taken up into the cell by endosomes, and is degraded in the cell to efficiently express the nucleic acid.

[0130] また、（-)鎖 RNAウィルスであるセンダイウィルスをベースにしたウィルスベクターも開 発されており（W097/16538、 W097/16539)、該センダイウィルスを用いて、本発明に 用いるマイクロ RNAまたはマイクロ RNA前駆体を組み込んだセンダイウィルスを作 製すること力 Sでさる。  [0130] In addition, viral vectors based on Sendai virus (-) strand RNA virus have been developed (W097 / 16538, W097 / 16539), and microRNAs used in the present invention can be obtained using the Sendai virus. Or, you can use S to produce a Sendai virus that incorporates a microRNA precursor.

本発明に用いられる核酸、配列番号 1〜201からなる塩基配列と相補的な塩基配 列を含む核酸、または、マイクロ RNAの標的遺伝子に対する siRNAは、非ウィルス核 酸移入法によっても移入することができる。例えば、リン酸カルシウム共沈法 [Virology ， 52， 456-467 (1973)； Science, 209， 1414-1422 (1980)]、マイクロインジェクション法 [P roc.Natl.Acad.Sci. USA, 77， 5399-5403 (1980) ; Proc.Natl.Acad.Sci. USA, 77， 7380- 7384 (1980) ; Cell, 27， 223-231 (1981) ; Nature, 294， 92-94 (1981)]、リボソームを介し た膜融合-介在移入法 [Proc.Natl.Acad.Sci. USA, 84， 7413-7417 (1987) ; Biochemis try, 28， 9508-9514 (1989) ; J. Biol. Chem., 264， 12126-12129 (1989) ; Hum. Gene Th er.， 3， 267-275， (1992) ; Science, 249， 1285-1288 (1990) ; Circulation, 83， 2007-201 1 (1992)]あるいは直接 DNA取り込みおよび受容体-媒介 DNA移入法 [Science, 247， 1465-1468 (1990) ; J. Biol. Chem., 266， 14338-14342 (1991) ; Proc.Natl.Acad.Sci. U SA, 87， 3655-3659 (1991) ; J. Biol. Chem., 264， 16985-16987 (1989) ; BioTechnique s， 11， 474-485 (1991) ; Proc.Natl.Acad.Sci. USA, 87， 3410-3414 (1990) ; Proc.Natl.A cad.Sci. USA, 88， 4255-4259 (1991) ; Proc.Natl.Acad.Sci. USA, 87， 4033-4037 (19 90) ; Proc.Natl.Acad.Sci. USA, 88， 8850-8854 (1991) ; Hum. Gene Ther., 3， 147-15 4 (1991)]等により移人することカできる。 The nucleic acid used in the present invention, a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201, or siRNA for a target gene of microRNA is a non-viral nucleus. It can also be transferred by the acid transfer method. For example, calcium phosphate coprecipitation method [Virology, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980); Proc.Natl.Acad.Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294, 92-94 (1981)], via ribosomes Membrane fusion-mediated transfer [Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J. Biol. Chem., 264, 12126-12129 (1989); Hum. Gene Ther., 3, 267-275, (1992); Science, 249, 1285-1288 (1990); Circulation, 83, 2007-201 1 (1992)] or direct DNA uptake and reception Body-mediated DNA transfer [Science, 247, 1465-1468 (1990); J. Biol. Chem., 266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655-3659 (1991); J. Biol. Chem., 264, 16985-16987 (1989); BioTechniques, 11, 474-485 (1991); Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990) ; Proc.Natl.A cad.Sci. USA, 88, 4255-4259 (1991); Proc.Natl.Acad.Sci. USA, 87, 4033-4037 (19 90); Proc.Natl.Acad.Sci. USA, 88, 8850-8854 (1991); Hum. Gene Ther ., 3, 147-15 4 (1991)].

[0131] リボソームを介した膜融合一介在移入法は、リボソーム調製物を目的とする組織に 直接投与することにより、本発明に用いられる核酸、配列番号 1〜201からなる塩基 配列と相補的な塩基配列を含む核酸、または、マイクロ RNAの標的遺伝子に対する siRNAを該組織の局所に取り込み、および発現させること力 Sできる [Hum. Gene Ther. ， 3， 399 (1992)]。 DNAを病巣に直接ターゲッティングするには、直接 DNA取り込み技 術が好ましい。 [0131] Ribosome-mediated membrane fusion-mediated transfer is a method in which a ribosome preparation is directly administered to a target tissue so that it is complementary to the nucleic acid used in the present invention, the base sequence consisting of SEQ ID NOs: 1 to 201. A nucleic acid containing a base sequence or siRNA for a target gene of microRNA can be taken up locally and expressed in the tissue [Hum. Gene Ther., 3, 399 (1992)]. Direct DNA uptake techniques are preferred for targeting DNA directly to the lesion.

[0132] 受容体-媒介 DNA移入は、例えば、ポリリジンを介して、蛋白質リガンドに DNA (通 常、共有的に閉環したスーパーコイル化プラスミドの形態をとる）を結合することによ つて行う方法をあげることができる。リガンドは、 目的細胞または組織の細胞表面上の 対応するリガンド受容体の存在に基づいて選択する。該リガンドー DNAコンジユゲー トは、所望により、血管に直接注射することができ、受容体結合および DNA—蛋白質 コンプレックスの内在化が起こる標的組織に指向し得る。 DNAの細胞内破壊を防止 するために、アデノウイルスを同時感染させて、エンドソーム機能を崩壊させることも できる。 11.聞 榦細朐から骨芽細朐,への分化および評価方法 [0132] Receptor-mediated DNA transfer is performed, for example, by binding DNA (typically in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. I can give you. The ligand is selected based on the presence of the corresponding ligand receptor on the cell surface of the cell or tissue of interest. The ligand-DNA complex can be injected directly into the blood vessel, if desired, and can be directed to a target tissue where receptor binding and internalization of the DNA-protein complex occurs. To prevent intracellular destruction of DNA, adenovirus can be co-infected to disrupt endosomal function. 11.Differentiation and evaluation method from 榦榦 細 朐 to osteoblast 朐

間葉系幹細胞から骨芽細胞への分化過程における影響を調べる方法としては、例 えば以下のようにして確認すること力 Sできる。具体的には、間葉系幹細胞から骨芽細 胞へ分化を誘導する条件下で、本発明で用いられる核酸、または、マイクロ RNAの 標的遺伝子に対する siRNAを間葉系幹細胞に導入し、培養する。骨芽細胞への分 化が進むに伴い発現が上昇する遺伝子または蛋白質を解析し、陰性対照と比較す  As a method for examining the influence in the differentiation process from mesenchymal stem cells to osteoblasts, for example, it is possible to confirm as follows. Specifically, under conditions that induce differentiation from mesenchymal stem cells to osteoblasts, the nucleic acid used in the present invention or siRNA for the target gene of microRNA is introduced into mesenchymal stem cells and cultured. . Analyze genes or proteins that increase in expression as they differentiate into osteoblasts and compare to negative controls

[0133] 間葉系幹細胞から骨芽細胞へ分化誘導させる方法としては、間葉系幹細胞から骨 芽細胞へ分化誘導させることができる方法であれば!/、かなる方法でもよ!/、が、例えば 、 Science, 284, 143-147 (1999)に記載の方法があげられる。具体的には、間葉系幹 細胞を培養器に播種した後、デキサメサゾン、ァスコルビン酸ー2リン酸、および /3— グリセ口フォスフェートを含む細胞培養用培地中で 1〜4週間培養を続けることにより 、間葉系幹細胞を骨芽細胞へ分化させることができる。 [0133] As a method for inducing differentiation from mesenchymal stem cells to osteoblasts, any method capable of inducing differentiation from mesenchymal stem cells to osteoblasts! /, Any method may be used! / For example, the method described in Science, 284, 143-147 (1999) can be mentioned. Specifically, after inoculating mesenchymal stem cells in an incubator, continue culturing for 1 to 4 weeks in a cell culture medium containing dexamethasone, ascorbic acid-2-phosphate, and / 3-glycose oral phosphate Thus, mesenchymal stem cells can be differentiated into osteoblasts.

[0134] 骨芽細胞への分化に伴い発現が変動する遺伝子の定量的な解析法としては、 RT- Pし R、reverse transcription— polymerase chain reactionノ、ノーザノノ、ィプリタ セーン ヨン、ドットブロットハイブリダィゼーシヨン、 DNAマイクロアレイ等を用いた方法があげ られる。  [0134] Quantitative analysis of genes whose expression varies with differentiation into osteoblasts includes RT-P, R, reverse transcription—polymerase chain reaction, nozano, yipryta, and dot blot hybridiy. Examples include a method using a seed or DNA microarray.

骨芽細胞への分化に伴い発現が変動する蛋白質の定量的な解析法としては、ゥェ スタンプロット解析、該蛋白質に特異的に反応する抗体を用いた免疫組織染色、 ELI SA等があげられる。  Quantitative analysis methods for proteins whose expression varies with osteoblast differentiation include Western blot analysis, immunohistochemical staining using antibodies that react specifically with the protein, and ELI SA. .

[0135] 骨芽細胞への分化に伴い発現が上昇する遺伝子または蛋白質としては、 I型コラー ゲン、ォステオカルシン、ォステオネタチン、ォステオポンチン、ボーンシァロプロティ ン（bone sialoprotein) , Runx2 (runt-related gene 2)、ァノレカジフォスファタ一T(ALP) 等があげられる。  [0135] Genes or proteins whose expression increases with differentiation into osteoblasts include type I collagen, osteocalcin, osteonetatin, osteopontin, bone sialoprotein, Runx2 (runt-related gene 2) Anoleka diphosphatase T (ALP) and the like.

また、骨芽細胞への分化程度を評価する方法としては、該骨芽細胞中の ALP酵素 活性を利用して細胞を染色する方法、あるいは、 ALP酵素活性を測定する方法があ げられる。該細胞染色のより具体的な方法は、骨芽細胞中の ALP酵素により加水分 解された基質のリン酸エステルのアルコール部分をジァゾニゥム塩でカップリングし、 ァゾ色素で酵素活性部位に沈殿させる方法をあげられる。基質としては、例えばナフ トーノレ AS-MXリン酸を、ァゾ色素としては、例えばファーストバイオレット青をあげるこ とができる。これらが含まれているキット、例えば leukocyte alkaline phosphatase (Sigm a社製）等を用いてもよい。また、 ALP酵素活性の測定キットとして、例えばアルカリ性 ホスファ B-テストヮコー（和光純薬株式会社製）等を用いてもよい。 Examples of methods for evaluating the degree of differentiation into osteoblasts include a method of staining cells using the ALP enzyme activity in the osteoblast, or a method of measuring ALP enzyme activity. A more specific method of the cell staining is to couple the alcohol portion of the phosphate ester of the substrate hydrolyzed by ALP enzyme in osteoblasts with a diazonium salt, A method of precipitating with an azo dye on the enzyme active site can be mentioned. As the substrate, for example, naphthonor AS-MX phosphoric acid can be mentioned, and as the azo dye, for example, first violet blue can be mentioned. A kit containing these may be used, such as leukocyte alkaline phosphatase (manufactured by Sigma). Further, as a kit for measuring ALP enzyme activity, for example, alkaline phospha B-test coco (manufactured by Wako Pure Chemical Industries, Ltd.) may be used.

[0136] 更に、骨芽細胞が産生した石灰化成分を検出することによつても骨芽細胞への分 化を確認することもできる。石灰化成分を検出する方法としては、フォンコッサ (von ossa)染色、ァリザリンレッド（Alizarin Red)染色等の染色法があげられる。 [0136] Furthermore, the differentiation into osteoblasts can also be confirmed by detecting the calcification component produced by the osteoblasts. Examples of the method for detecting the calcified component include staining methods such as von ossa staining and alizarin red staining.

フォンコッサ染色とは、硝酸銀を用いて石灰化成分であるリン酸カルシウムを検出 する方法である。具体的には、パラフィン等で固定した細胞に対し 1〜5%の硝酸銀 水溶液を反応させ光に当て、黒く呈色したリン酸カルシウムが存在する部分を定量、 例えば該呈色面積を計測することにより骨芽細胞への分化程度を評価することがで きる。  Von Kossa staining is a method of detecting calcium phosphate, which is a calcifying component, using silver nitrate. Specifically, the cells fixed with paraffin or the like are reacted with 1 to 5% silver nitrate aqueous solution and exposed to light, and the portion where the black calcium phosphate is present is quantified, for example, the bone area is measured by measuring the colored area. The degree of differentiation into blast cells can be evaluated.

[0137] ァリザリンレッド染色とは、ァリザリン赤 Sがカルシウムに対して特異的な結合を示し レーキを形成することを利用した方法である。具体的には、パラフィン等で固定した細 胞に対し 0.01〜5%ァリザリン赤 S溶液を反応させる、赤紫色〜橙赤色に呈色した部 分を定量、例えば該呈色面積を計測することにより骨芽細胞への分化程度を評価す ること力 Sでさる。  [0137] Alizarin red staining is a method utilizing the fact that alizarin red S shows a specific binding to calcium and forms a rake. Specifically, by reacting 0.01 to 5% alizarin red S solution with cells fixed with paraffin or the like, the colored portion of reddish purple to orange red is quantified, for example, by measuring the colored area. Ability to evaluate the degree of differentiation into osteoblasts.

[0138] 以下、実施例により本発明を具体的に説明する。  [0138] The present invention will be specifically described below with reference to examples.

実施例 1  Example 1

[0139] 間葉系幹細胞で発現するマイクロ RNAの単離  [0139] Isolation of microRNA expressed in mesenchymal stem cells

(1) RNA抽出  (1) RNA extraction

ヒト間葉系幹細胞（以下、 hMSCと称す）は Cambrex社より入手し、 20%ゥシ胎児血清 (FBS) (JRH Bioscience社製）を含む IMDM培地（Invitrogen社製）で 37°Cの 5%C02 濃度のインキュベータ一中で培養した。培養中の hMSCから TRIZOL reagent (Invitro gen社製）を用いて、製品に添付された方法に従い全 RNAを抽出した。  Human mesenchymal stem cells (hereinafter referred to as hMSC) were obtained from Cambrex, and IMDM medium (Invitrogen) containing 20% urine fetal serum (FBS) (manufactured by JRH Bioscience) was 5% at 37 ° C. The cells were cultured in a C02 concentration incubator. Total RNA was extracted from hMSC in culture using TRIZOL reagent (Invitrogen) according to the method attached to the product.

(2)低分子 RNAのクローニング  (2) Cloning of small RNA

上記（1)で取得した hMSC由来全 RNAZOO ^ gを用いて、 Lauらの方法（Science 294， 858-862， 2001)に従い、低分子 RNAのクローニングを行なった。具体的には、 15%ポ リアクリルアミドゲル電気泳動後、低分子 RNAを切り出し、該低分子 RNAに対して 5' アデニル化 3'—アダプターライゲーシヨン、 5' アダプターライゲーシヨン、逆転写 、 PCR増幅、コンカテマ一化、 pCR2.1-TOPOベクターへのライゲーシヨンを順次進め 、クローユングした低分子 RNAの塩基配列を決定した。なお 5'—アデニル化 3'—ァ ダプターは、 Integrated DNA Technologies社製の miRNA Cloning Linkerを用いた。 Using the hMSC-derived total RNAZOO ^ g obtained in (1) above, the method of Lau et al. (Science 294, 858-862, 2001), low molecular RNA was cloned. Specifically, after 15% polyacrylamide gel electrophoresis, low molecular RNA was excised, and 5 ′ adenylation 3′-adapter ligation, 5 ′ adapter ligation, reverse transcription, PCR amplification, concatamerization, and ligation to the pCR2.1-TOPO vector were sequentially performed, and the base sequence of the cloned small RNA was determined. As the 5′-adenylation 3′-adapter, miRNA Cloning Linker manufactured by Integrated DNA Technologies was used.

(3)マイクロ RNAの同定  (3) Identification of microRNA

上記（2)で決定した塩基配列を、マイクロ RNAデータベースである miRBase (http:/ /microrna.sanger.ac.uk/)に登録されている塩基配列と比較することにより、間葉系 幹細胞で発現するマイクロ RNAを選択した。このようにして選択した間葉系幹細胞で 発現するマイクロ RNAとしては、例えば、配列番号 1 (hsa-miR-24)、配列番号 2 (hsa_ miR-100)、配列番号 3 (hsa-miR-145)、配列番号 4 (hsa-miR-210)、配列番号 5 (hsa -miR-422b)で表されるマイクロ RNAを挙げることができる。  Expressed in mesenchymal stem cells by comparing the base sequence determined in (2) above with the base sequence registered in miRBase (http://microrna.sanger.ac.uk/), a microRNA database The microRNA to be selected was selected. Examples of microRNAs expressed in mesenchymal stem cells selected in this way include, for example, SEQ ID NO: 1 (hsa-miR-24), SEQ ID NO: 2 (hsa_miR-100), SEQ ID NO: 3 (hsa-miR-145). ), SEQ ID NO: 4 (hsa-miR-210) and SEQ ID NO: 5 (hsa-miR-422b).

実施例 2  Example 2

[0140] マイクロ RNAを強制発現した間葉系幹細胞の骨芽細胞分化  [0140] Osteoblast differentiation of mesenchymal stem cells forcibly expressing microRNA

実施例 1で得られたマイクロ RNAの前駆体を hMSCに導入し、骨芽細胞へ分化誘導 をおこなう条件下で培養し、該マイクロ RNA前駆体の影響を調べた。  The microRNA precursor obtained in Example 1 was introduced into hMSC, cultured under conditions for inducing differentiation into osteoblasts, and the influence of the microRNA precursor was examined.

hMSCを 24穴プレートに 1穴あたり 6·2χ103個になるように播種し、 20% FBSを含む Μ DM培地でー晚培養した。 1日後、マイクロ RNA前駆体をリポフエクシヨン法、具体的に は、 Lipofectamine2000 (Invitrogen社製）を用いて、終濃度力 ¾0nMとなるよう hMSCに 導入した。マイクロ RNAの前駆体としては、 Ambion社の Pre-miR™miRNA Precursor Moleculesを用い、リポフエクシヨン法は、製品に添付された方法に従った。 hMSCs were seeded in a 24-well plate so that there were 6 · 2 × 10 3 per well, and cultured in a DM medium containing 20% FBS. One day later, the microRNA precursor was introduced into the hMSC using a lipofection method, specifically, Lipofectamine 2000 (manufactured by Invitrogen) to a final concentration force of ¾0 nM. As a microRNA precursor, Ambion's Pre-miR ™ miRNA Precursor Molecules was used, and the lipofusion method was in accordance with the method attached to the product.

[0141] リボフヱクシヨン法により該マイクロ RNA前駆体を導入した 1日後、培地を骨芽分化 誘導培地 [20%FBSを含む IMDM培地中に、 0.1 ^ mol/Lデキサメサゾン、 50 ^ mol/L ァスコルビン酸ー2リン酸（Sigma社製）、 10mmol/L /3—グリセ口フォスフイト（Sigma社 製) ]へ交換し、 3日に一度の頻度で、骨芽分化誘導培地を交換して培養を続けた。 培養開始から 2週間後に、細胞の形態を位相差顕微鏡 (Nikon社製）下で観察し、 更にアルカリフォスファターゼ染色を行って、骨芽細胞を検出した。具体的には、ま ず、細胞をリン酸緩衝液（phosphate-buffered saline： PBS) (Invitrogen社製）で 1回洗 浄し、固定液（10% formalin/PBS)で 5分間固定した。蒸留水で洗浄した後、喑所で N aphthol AS-MXリン酸（Sigma社製）と Fast Violet B溶液との混合溶液（Sigma社製）と 3 0分間反応させ、アルカリフォスファターゼ反応をおこなった。更に蒸留水で洗浄し、 位相差顕微鏡下で赤く染まって!/、る骨芽細胞を観察し、デジタルカメラ (Nikon社製） で撮影した。 [0141] One day after the introduction of the microRNA precursor by the ribofusion method, the medium was induced to induce osteoblast differentiation [in IMDM medium containing 20% FBS, 0.1 ^ mol / L dexamethasone, 50 ^ mol / L ascorbic acid- 2 phosphoric acid (manufactured by Sigma), 10 mmol / L / 3-glyce mouth phosphate (manufactured by Sigma)], and the osteoblast differentiation induction medium was replaced once every three days to continue the culture. Two weeks after the start of culture, the cell morphology was observed under a phase contrast microscope (Nikon), and further, alkaline phosphatase staining was performed to detect osteoblasts. Specifically, First, the cells were washed once with phosphate-buffered saline (PBS) (manufactured by Invitrogen) and fixed with fixative (10% formalin / PBS) for 5 minutes. After washing with distilled water, the mixture was reacted with a mixed solution of Naphthol AS-MX phosphate (manufactured by Sigma) and Fast Violet B solution (manufactured by Sigma) for 30 minutes to perform an alkaline phosphatase reaction. Further, it was washed with distilled water, stained red under a phase contrast microscope! /, And the osteoblasts were observed and photographed with a digital camera (Nikon).

[0142] その結果、 hsa- miR- 24、 hsa-miR- 100、 hsa- miR- 145、 hsa- miR- 210、 hsa-miR- 422b の前駆体を導入した hMSCは、マイクロ RNA前駆体非導入 hMSCと比べて、細胞数が 少なぐアルカリフォスファターゼ染色された陽性細胞の数も少ないことが見出された 。マイクロ RNA前駆体は細胞中でマイクロ RNAに変換されるので、本実施例で用いた マイクロ RNAは、 hMSCに対して、増殖を抑制する活性および骨芽細胞への分化を抑 制する活性を有することがわ力、つた。  [0142] As a result, hMSCs with hsa-miR-24, hsa-miR-100, hsa-miR-145, hsa-miR-210, and hsa-miR-422b precursors were introduced without microRNA precursors. It was found that the number of positive cells stained with alkaline phosphatase was smaller than that of hMSC. Since the microRNA precursor is converted into microRNA in the cell, the microRNA used in this example has an activity to suppress proliferation and an activity to suppress differentiation into osteoblasts against hMSC. Kotawa power.

実施例 3  Example 3

[0143] 間葉系幹細胞が骨芽細胞へ分化している渦程におけるマイクロ RNAの発現量解析  [0143] Expression analysis of microRNA in the vortex of mesenchymal stem cells differentiated into osteoblasts

実施例 2で hMSCの増殖および分化抑制活性を示したマイクロ RNAにつ!/、て、 hMS For microRNAs that showed hMSC proliferation and differentiation-inhibiting activity in Example 2! /, HMS

Cの骨芽細胞分化過程の発現量変動を、定量的 PCRを用いて調べた。 Changes in the expression level of C osteoblast differentiation were examined using quantitative PCR.

hMSCを上記の骨芽分化誘導培地で培養し、培養開始から 1日後、 3日後、 5日後、 hMSC is cultured in the above osteoblast differentiation medium, 1 day, 3 days, 5 days after the start of culture,

7日後、 10日後、 14日後、 21日後および対照として、分化誘導前の hMSCから、それ ぞれ全 RNAを抽出した。全 RNAは、 mirVana miRNA Isolation Kit (Ambion社製）を用 い、製品に添付された方法に従った。 Total RNA was extracted from hMSCs before differentiation induction at 7 days, 10 days, 14 days, 21 days and as a control. For the total RNA, mirVana miRNA Isolation Kit (Ambion) was used and the method attached to the product was followed.

[0144] 抽出した全 RNAlOngを用いて、 TaqMan MicroRNA Assays (アプライドバイオシステ ムズ社製）を用いて添付の方法に従い反応させ、 AB画 S列検出システム（ABI PRISM[0144] Using the extracted total RNAlOng, TaqMan MicroRNA Assays (manufactured by Applied Biosystems) is reacted according to the attached method, and the AB column S row detection system (ABI PRISM

7700、アプライドバイオシステムズ社製）を用いてリアルタイム PCRを行った。 Real-time PCR was performed using 7700 (Applied Biosystems).

発現量は、シグナル強度が 1000に達する時のサイクル数 (Ct値)を求めた後、ネガテ イブコントロールの Ct値から上記値を引き、 A Ct値で計算した。なお、ネガティブコン トロールは、滅菌水を用いた PCRの Ct値を利用した。  The expression level was calculated as the A Ct value after obtaining the number of cycles (Ct value) when the signal intensity reached 1000 and then subtracting the above value from the negative control Ct value. Negative control used the Ct value of PCR using sterilized water.

[0145] 分化誘導前の hMSCの発現量を 1. 0として、それぞれの相対発現量を計算した。そ の結果、図 1に示したように、どのマイクロ RNAも骨芽分化誘導時の初期は、発現量 に大きな増加は見られな力、つた。 [0145] The expression level of hMSC before differentiation induction was 1.0, and the relative expression level of each was calculated. As a result, as shown in Fig. 1, the expression level of any microRNA at the initial stage of osteoblast differentiation was There has been no significant increase in power.

実施例 4  Example 4

[0146] hsa-miR-145の予測標白 眚ィ云早# 糸田朐 に すろ醫響  [0146] Prediction of hsa-miR-145

実施例 2で hMSCの増殖および分化抑制活性を示した hsa-miR-145の標的遺伝子 候補を検索し、 ADD3遺伝子および CBFB遺伝子の siRNAを hMSCに導入して、該遺 伝子の発現を抑制した条件下における hMSCが骨芽細胞へ分化する過程を調べた。 実施例 2と同様の方法で、 hMSCを骨芽分化誘導培地中で培養した。  Searched for hsa-miR-145 target gene candidates that showed hMSC proliferation and differentiation suppression activity in Example 2, and introduced siRNAs of ADD3 gene and CBFB gene into hMSC to suppress the expression of the gene The process of hMSC differentiation into osteoblasts under the conditions was investigated. In the same manner as in Example 2, hMSCs were cultured in an osteoblast differentiation induction medium.

[0147] ADD3遺伝子（配列番号 206)の発現を抑制する siRNAとしては、配列番号 202で 示される塩基配列を標的とする siRNA、および配列番号 203で示される塩基配列を 標的とする siRNAを化学合成した。 CBFB遺伝子（配列番号 207)の発現を抑制する si RNAとしては配列番号 204で示される塩基配列を標的とする siRNA、および配列番 号 205で示される塩基配列を標的とする siRNAを化学合成した。化学合成した 2重鎖 RNAをリポフエクシヨン法により hMSCに導入した。  [0147] As siRNAs that suppress the expression of the ADD3 gene (SEQ ID NO: 206), the siRNA targeting the nucleotide sequence represented by SEQ ID NO: 202 and the siRNA targeting the nucleotide sequence represented by SEQ ID NO: 203 are chemically synthesized. did. As siRNAs that suppress the expression of the CBFB gene (SEQ ID NO: 207), siRNA targeting the nucleotide sequence represented by SEQ ID NO: 204 and siRNA targeting the nucleotide sequence represented by SEQ ID NO: 205 were chemically synthesized. The chemically synthesized double-stranded RNA was introduced into hMSC by the lipofusion method.

[0148] 該 2重鎖 RNAを導入してから 2週後、細胞の形態を位相差顕微鏡 (Nikon社製）下で 観察し、更にアルカリフォスファターゼ染色により赤く染まっている骨芽細胞をデジタ ノレカメラ (Nikon社製）で撮影した。  [0148] Two weeks after the introduction of the double-stranded RNA, the morphology of the cells was observed under a phase-contrast microscope (Nikon), and osteoblasts stained in red by alkaline phosphatase staining were further detected with a digital camera ( Nikon).

その結果、 ADD3遺伝子の siRNAを導入した hMSCにおいては、 siRNA非導入 hMSC に比べて、細胞数が少なぐアルカリフォスファターゼ染色された陽性細胞の数が少 ないことが示された。つまり、 ADD3遺伝子の機能を抑制すると、 hMSCの増殖および 骨芽細胞への分化が抑制された。また、 CBFB遺伝子の siRNAを導入した hMSCにお いては、 siRNA非導入 hMSCと比べて、アルカリフォスファターゼ染色された陽性細胞 の数が少ないことが示された。以上より CBFB遺伝子の機能を抑制すると、 hMSCの骨 芽細胞への分化が抑制されることが示された。  As a result, it was shown that the number of positive cells stained with alkaline phosphatase was smaller in hMSCs into which siRNA of the ADD3 gene was introduced, compared to hMSCs into which siRNA had not been introduced. In other words, suppression of ADD3 gene function suppressed hMSC proliferation and differentiation into osteoblasts. It was also shown that the number of positive cells stained with alkaline phosphatase was smaller in hMSCs into which siRNA of the CBFB gene was introduced, compared to hMSCs into which no siRNA had been introduced. These results indicate that hMSC differentiation into osteoblasts is suppressed when CBFB gene function is suppressed.

実施例 5  Example 5

[0149] hsa-miR-145の標的遺伝子がコードする蛋白晳の発現量解析  [0149] Analysis of the expression level of the protein encoded by the target gene of hsa-miR-145

実施例 4で hMSCの増殖や分化に対して抑制活性を示した hsa-miR-145前駆体を h MSCへ導入し、該標的遺伝子がコードする蛋白質量の変化を調べた。  The hsa-miR-145 precursor that showed inhibitory activity against hMSC proliferation and differentiation in Example 4 was introduced into hMSC, and changes in the amount of protein encoded by the target gene were examined.

Ambion社の hsa-miR-145前駆体を終濃度 2nMあるいは 20nMとなるように Lipofectam ine2000を用いて予め播種していた 1.2xl05個の hMSCに導入した。導入 3日後に、細 胞を Lysis buffer[50mM Tris— HCL(pH7.4)、 150mM NaCl、 ImM EDTA、 1% Nonident P-40、 1% Protease Inhibitor Cocktail SetIII (Calbiochem社製）]に溶解して回収した。 定法により SDS-PAGE法で分離した後、ウェスタンブロッテイング法で、 ADD3および C BFBの蛋白量を検出し、定量した。 ADD3の検出には、抗 ADD3抗体（SantaCruz社製 )を、 CBFBの検出には抗 CBFB抗体（Abeam社製）を用いた。 Lipofectam with Ambion hsa-miR-145 precursor to a final concentration of 2nM or 20nM It was introduced into 1.2 x15 5 hMSCs previously seeded with ine2000. Three days after introduction, the cells were dissolved in Lysis buffer [50 mM Tris—HCL (pH 7.4), 150 mM NaCl, ImM EDTA, 1% Nonident P-40, 1% Protease Inhibitor Cocktail Set III (Calbiochem)]. It was collected. After separation by SDS-PAGE by a conventional method, the amounts of ADD3 and CBFB proteins were detected and quantified by Western blotting. Anti-ADD3 antibody (manufactured by SantaCruz) was used for detection of ADD3, and anti-CBFB antibody (manufactured by Abeam) was used for detection of CBFB.

[0150] hsa-miR-145前駆体を強制発現した hMSCは、陰性コントロールの hMSCと比較して Addcin3および CBFBの蛋白量が減少したので、 ADD3および CBFB遺伝子は、 hsa_m iR-145の標的遺伝子であり、発現が調節されていることが示された。 [0150] The hMSCs that forcibly expressed the hsa-miR-145 precursor had decreased amounts of Addcin3 and CBFB proteins compared to the negative control hMSC, so the ADD3 and CBFB genes were the target genes of hsa_m iR-145. It was shown that expression is regulated.

産業上の利用可能性  Industrial applicability

[0151] 本発明のマイクロ RNAなどの核酸やその誘導体は、間葉系幹細胞の増殖および /または分化抑制、マイクロ RNAの標的遺伝子の発現抑制、間葉系幹細胞の増殖 および/または分化の異常に起因する疾患の診断、治療などに有用である。 [0151] A nucleic acid such as microRNA or a derivative thereof of the present invention is effective in suppressing the proliferation and / or differentiation of mesenchymal stem cells, suppressing the expression of a target gene of microRNA, and abnormally growing and / or differentiating mesenchymal stem cells. It is useful for diagnosis and treatment of diseases caused by it.

Claims

請求の範囲 The scope of the claims [1] 以下の（a)〜 (j)のいずれかの核酸を有効成分として含有する間葉系幹細胞の増殖 および/または分化抑制剤。  [1] A mesenchymal stem cell proliferation and / or differentiation inhibitor containing any of the following nucleic acids (a) to (j) as an active ingredient. (a)配列番号;!〜 93のいずれかで表される塩基配列からなる核酸  (a) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs:! to 93 (b)配列番号 1〜93のいずれかで表される塩基配列を含有する、 15〜28塩基の核 酸  (b) a 15-28 base nucleic acid containing the base sequence represented by any of SEQ ID NOs: 1-93 (c)配列番号 1〜93のいずれかで表される塩基配列 90%以上の同一性を有する塩 基配列からなる核酸  (c) a nucleic acid comprising a base sequence having 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 93 (d)配列番号 1〜93のいずれかで表される塩基配列からなる核酸の相補鎖とストリン ジェントな条件でハイブリダィズする核酸  (d) a nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid comprising the nucleotide sequence represented by any of SEQ ID NOs: 1 to 93 (e)配列番号;!〜 5のいずれかで表される塩基配列の 2〜8番目の塩基配列を含む 核酸  (e) a nucleic acid comprising the second to eighth base sequences of the base sequence represented by any of SEQ ID NOs:! to 5 (f) (a)〜（e)のいずれかの核酸と、該核酸の塩基配列に対して相補的な塩基配列と からなる核酸とを含む二本鎖核酸  (f) A double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid (g) (a)〜（e)のいずれかの核酸と、該核酸とストリンジェントな条件でハイブリダィズ する核酸とからなる二本鎖核酸  (g) Double-stranded nucleic acid comprising the nucleic acid of any one of (a) to (e) and a nucleic acid that hybridizes with the nucleic acid under stringent conditions (h)配列番号 94〜201のいずれかで表される塩基配列からなる核酸  (h) a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201 (i)配列番号 94〜201のいずれかで表される塩基配列と 90%以上の同一性を有す る塩基配列からなる核酸  (i) a nucleic acid comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence represented by any of SEQ ID NOS: 94 to 201 (j)配列番号 94〜201のいずれかで表される塩基配列からなる核酸の相補鎖とストリ ンジェントな条件でハイブリダィズする核酸。  (j) A nucleic acid that hybridizes with a complementary strand of a nucleic acid comprising the base sequence represented by any of SEQ ID NOS: 94 to 201 under stringent conditions. [2] 請求項 1に記載の核酸を発現するベクターを有効成分として含有する間葉系幹細胞 の増殖および/または分化抑制剤。 [2] A mesenchymal stem cell proliferation and / or differentiation inhibitor containing the nucleic acid-expressing vector of claim 1 as an active ingredient. [3] 請求項 1に記載の核酸の標的遺伝子の発現を抑制する物質を有効成分として含有 する間葉系幹細胞の増殖および/または分化抑制剤。 [3] A mesenchymal stem cell proliferation and / or differentiation inhibitor comprising a substance that suppresses the expression of the target gene of the nucleic acid according to claim 1 as an active ingredient. [4] ADD3遺伝子または CBFB遺伝子の発現を抑制する物質を有効成分として含有す る間葉系幹細胞の増殖および/または分化抑制剤。 [4] A mesenchymal stem cell proliferation and / or differentiation inhibitor containing a substance that suppresses the expression of ADD3 gene or CBFB gene as an active ingredient. [5] ADD3遺伝子または CBFB遺伝子の発現を抑制する物質が核酸である請求項 4に 記載の間葉系幹細胞の増殖および/または分化抑制剤。 [5] The substance according to claim 4, wherein the substance that suppresses the expression of ADD3 gene or CBFB gene is a nucleic acid. The described mesenchymal stem cell proliferation and / or differentiation inhibitor. [6] 核酸が siRNAである請求項 5に記載の間葉系幹細胞の増殖および/または分化抑 制剤。 6. The agent for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to claim 5, wherein the nucleic acid is siRNA. [7] siRNAが配列番号 202〜205の!/、ずれかで表される塩基配列を標的配列とする siR NAである請求項 6記載の間葉系幹細胞の増殖および/または分化抑制剤。  7. The agent for suppressing proliferation and / or differentiation of mesenchymal stem cells according to claim 6, wherein the siRNA is siRNA having a target sequence of the nucleotide sequence represented by SEQ ID NO: 202 to 205! [8] 請求項 1に記載の核酸を有効成分として含有する、該核酸の標的遺伝子の発現抑 制剤。  [8] An expression inhibitor for a target gene of the nucleic acid, comprising the nucleic acid according to claim 1 as an active ingredient. [9] 配列番号 3で表される塩基配列からなる核酸または配列番号 97で表される塩基配列 力 なる核酸を有効成分とする ADD3遺伝子の発現抑制剤。  [9] An ADD3 gene expression inhibitor comprising as an active ingredient a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 97. [10] 配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基配列 とからなる核酸とを含む二本鎖核酸を有効成分として含有する ADD3遺伝子の発現 抑制剤。 [10] ADD3 gene expression inhibitor comprising as an active ingredient a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid . [11] 配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズする核 酸とからなる二本鎖核酸を有効成分として含有する ADD3遺伝子の発現抑制剤。  [11] An ADD3 gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising a nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions. [12] 配列番号 3で表される塩基配列からなる核酸または配列番号 97で表される塩基配列 力 なる核酸を有効成分とする CBFB遺伝子の発現抑制剤。 [12] A CBFB gene expression inhibitor comprising as an active ingredient a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the base sequence represented by SEQ ID NO: 97. [13] 配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基配列 とからなる核酸とを含む二本鎖核酸を有効成分として含有する CBFB遺伝子の発現 抑制剤。 [13] CBFB gene expression inhibitor containing, as an active ingredient, a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid . [14] 配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズする核 酸とからなる二本鎖核酸を有効成分として含有する CBFB遺伝子の発現抑制剤。  [14] A CBFB gene expression inhibitor comprising, as an active ingredient, a double-stranded nucleic acid comprising a nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions. [15] 請求項 1記載の核酸を用いる、間葉系幹細胞の増殖および/または分化抑制方法。 [15] A method for inhibiting the proliferation and / or differentiation of mesenchymal stem cells using the nucleic acid according to claim 1. [16] 請求項 1に記載の核酸を発現するベクターを用いる、間葉系幹細胞の増殖および/ または分化抑制方法。 [16] A method for inhibiting the proliferation and / or differentiation of mesenchymal stem cells using the vector expressing the nucleic acid according to claim 1. [17] 請求項 1に記載の核酸の標的遺伝子の発現を抑制する物質を用いる間葉系幹細胞 の増殖および/または分化抑制方法。  [17] A method for suppressing proliferation and / or differentiation of mesenchymal stem cells using the substance that suppresses the expression of the target gene of the nucleic acid according to claim 1. [18] ADD3遺伝子または CBFB遺伝子の発現を抑制する物質を用いる間葉系幹細胞の 増殖および/または分化抑制方法。 [18] A method for suppressing proliferation and / or differentiation of mesenchymal stem cells using a substance that suppresses the expression of ADD3 gene or CBFB gene. [19] ADD3遺伝子または CBFB遺伝子の発現を抑制する物質が核酸である請求項 18 記載の間葉系幹細胞の増殖および/または分化抑制方法。 [19] The method for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to claim 18, wherein the substance that suppresses the expression of ADD3 gene or CBFB gene is a nucleic acid. [20] 核酸が siRNAである請求項 19記載の間葉系幹細胞の増殖および/または分化抑 制方法。 20. The method for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to claim 19, wherein the nucleic acid is siRNA. [21] siRNAが配列番号 202〜205のいずれかで表される塩基配列を標的配列とする siR [21] siR whose target sequence is the base sequence represented by any of SEQ ID NOS: 202 to 205 in siRNA NAである請求項 20記載の間葉系幹細胞の増殖および/または分化抑制方法。 21. The method for inhibiting proliferation and / or differentiation of mesenchymal stem cells according to claim 20, which is NA. [22] 請求項 1に記載の核酸を用いる、該核酸の標的遺伝子の発現抑制方法。 [22] A method for suppressing the expression of a target gene of the nucleic acid using the nucleic acid according to claim 1. [23] 配列番号 3で表される塩基配列からなる核酸、または配列番号 97で表される塩基配 列のからなる核酸を用いる ADD3遺伝子の発現抑制方法。 [23] A method for inhibiting ADD3 gene expression, which uses a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 97. [24] 配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基配列 とからなる核酸とを含む二本鎖核酸を用いる ADD3遺伝子の発現抑制方法。 [24] A method for suppressing ADD3 gene expression, comprising using a double-stranded nucleic acid comprising a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid. [25] 配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズする核 酸とからなる二本鎖核酸を用いる ADD3遺伝子の発現抑制方法。 [25] A method for inhibiting ADD3 gene expression, comprising using a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions. [26] 配列番号 3で表される塩基配列からなる核酸、または配列番号 97で表される塩基配 歹 IJからなる核酸を用いる CBFB遺伝子の発現抑制方法。 [26] A method for suppressing the expression of a CBFB gene using a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3 or a nucleic acid comprising the nucleotide sequence IJ represented by SEQ ID NO: 97. [27] 配列番号 3で表される塩基配列からなる核酸と、該核酸に対して相補的な塩基配列 とからなる核酸とを含む二本鎖核酸を用いる CBFB遺伝子の発現抑制方法。 [27] A method for inhibiting the expression of a CBFB gene using a double-stranded nucleic acid comprising a nucleic acid comprising a base sequence represented by SEQ ID NO: 3 and a nucleic acid comprising a base sequence complementary to the nucleic acid. [28] 配列番号 3で表される核酸と、該核酸とストリンジェントな条件でハイブリダィズする核 酸とからなる二本鎖核酸を用いる CBFB遺伝子の発現抑制方法。 [28] A method for inhibiting the expression of a CBFB gene using a double-stranded nucleic acid comprising the nucleic acid represented by SEQ ID NO: 3 and a nucleic acid that hybridizes with the nucleic acid under stringent conditions. [29] 請求項 1に記載の核酸の発現を促進させることを指標とする間葉系幹細胞の増殖お よび/または分化抑制剤のスクリーニング方法。 [29] A screening method for an agent for inhibiting the proliferation and / or differentiation of mesenchymal stem cells using the expression of the nucleic acid according to claim 1 as an index. [30] 請求項 1記載の核酸の標的遺伝子の発現を抑制させることを指標とする間葉系幹細 胞の増殖および/または分化抑制剤のスクリーニング方法。 [30] A method for screening an agent for suppressing proliferation and / or differentiation of a mesenchymal stem cell, using as an index suppression of expression of a target gene of the nucleic acid according to claim 1. [31] 請求項 1に記載の核酸の発現を抑制させることを指標とする間葉系幹細胞の増殖お よび/または分化促進剤のスクリーニング方法。 [31] A method for screening an agent for promoting the proliferation and / or differentiation of mesenchymal stem cells using the expression of the nucleic acid according to claim 1 as an index. [32] 請求項 1記載の核酸の標的遺伝子の発現を促進させることを指標とする間葉系幹細 胞の増殖および/または分化促進剤のスクリーニング方法。 [32] A method for screening an agent for promoting proliferation and / or differentiation of a mesenchymal stem cell using the expression of the target gene of the nucleic acid according to claim 1 as an index. [33] 請求項 1記載の核酸の発現を抑制させる物質を有効成分として含有する間葉系幹細 胞の増殖および/または分化促進剤。 [33] A mesenchymal stem cell comprising a substance that suppresses the expression of the nucleic acid according to claim 1 as an active ingredient Cell growth and / or differentiation promoter. [34] 配列番号 1〜201からなる塩基配列と相補的な塩基配列を含む核酸を有効成分とし て含有する間葉系幹細胞の増殖および/または分化促進剤。 [34] A mesenchymal stem cell proliferation and / or differentiation promoter containing, as an active ingredient, a nucleic acid containing a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201. [35] 請求項 1記載の核酸の発現を抑制させる物質を用いる間葉系幹細胞の増殖および[35] Proliferation of mesenchymal stem cells using a substance that suppresses the expression of the nucleic acid according to claim 1, and /または分化促進方法。 / Or differentiation promotion method. [36] 配列番号 1〜201からなる塩基配列と相補的な塩基配列を含む核酸を用いる間葉系 幹細胞の増殖および/または分化促進方法。 [36] A method for promoting proliferation and / or differentiation of mesenchymal stem cells using a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201. [37] 請求項 1に記載の核酸、または配列番号 1〜201からなる塩基配列と相補的な塩基 配列を含む核酸を有効成分として含有する、間葉系幹細胞の増殖および/または 分化の異常に起因する疾患の診断薬。 [37] An abnormality in proliferation and / or differentiation of mesenchymal stem cells, containing as an active ingredient the nucleic acid according to claim 1 or a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201 A diagnostic agent for the disease caused. [38] 請求項 1に記載の核酸の発現、該核酸の変異、該核酸をコードするゲノムの変異を 検出する試薬を有効成分として含有する、間葉系幹細胞の増殖および/または分化 の異常に起因する疾患の診断薬。 [38] Abnormality of proliferation and / or differentiation of mesenchymal stem cells, containing as an active ingredient a reagent for detecting the expression of the nucleic acid according to claim 1, mutation of the nucleic acid, or mutation of the genome encoding the nucleic acid A diagnostic agent for the disease caused. [39] 請求項 1に記載の核酸、または配列番号 1〜201からなる塩基配列と相補的な塩基 配列を含む核酸を有効成分として含有する、間葉系幹細胞の増殖および/または 分化の異常に起因する疾患の治療薬。 [39] An abnormality in proliferation and / or differentiation of mesenchymal stem cells, containing as an active ingredient the nucleic acid according to claim 1 or a nucleic acid comprising a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1 to 201 A treatment for the disease caused. [40] 請求項 1〜7のいずれかに記載の間葉系幹細胞の増殖および/または分化抑制剤 または請求項 33または 34に記載の間葉系幹細胞の増殖および/または分化促進 剤を有効成分として含有する間葉系幹細胞の増殖および/または分化の異常に起 因する疾患の治療薬。 [40] The mesenchymal stem cell proliferation and / or differentiation inhibitor according to any one of claims 1 to 7 or the mesenchymal stem cell proliferation and / or differentiation promoter according to claim 33 or 34 as an active ingredient A therapeutic agent for diseases caused by abnormal proliferation and / or differentiation of mesenchymal stem cells.
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