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WO2008053909A1 - Agent destiné à réguler la croissance et/ou la différenciation d'une cellule souche mésenchymateuse - Google Patents

Agent destiné à réguler la croissance et/ou la différenciation d'une cellule souche mésenchymateuse 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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Prior art keywords
nucleic acid
seq
differentiation
mesenchymal stem
base sequence
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PCT/JP2007/071177
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English (en)
Japanese (ja)
Inventor
Yoji Yamada
Tatsuya Miyazawa
Tetsuo Yoshida
Original Assignee
Kyowa Hakko Kirin Co., Ltd.
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Publication of WO2008053909A1 publication Critical patent/WO2008053909A1/fr

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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

L'invention concerne : un micro ARN ou un dérivé de celui-ci; un précurseur de micro ARN ou un dérivé de celui-ci; un acide nucléique à double brin composé du micro ARN ou de son dérivé et un acide nucléique comprenant une séquence de nucléotides complémentaire d'une séquence de nucléotides du micro ARN ou de son dérivé; un inhibiteur de la croissance et/ou de la différenciation d'une cellule souche mésenchymateuse qui comprend un acide nucléique à double brin composé du micro ARN ou de son dérivé et un acide nucléique capable de former des hybrides avec le micro ARN ou son dérivé dans des conditions rigoureuses; un inhibiteur de l'expression d'un gène cible du micro ARN; une utilisation de l'inhibiteur; et un agent diagnostique ou thérapeutique pour une maladie induite par une anomalie de la croissance et/ou de la différenciation d'une cellule souche mésenchymateuse.
PCT/JP2007/071177 2006-10-31 2007-10-31 Agent destiné à réguler la croissance et/ou la différenciation d'une cellule souche mésenchymateuse WO2008053909A1 (fr)

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* Cited by examiner, † Cited by third party
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JP2012509865A (ja) * 2008-11-26 2012-04-26 サントル ナシオナル ドゥ ラ ルシェルシェサイアンティフィク（セエヌエールエス） レトロウィルス感染を処理するための組成物及び方法
EP2440660A2 (fr) * 2009-06-10 2012-04-18 Chaya Brodie Procédés, systèmes et compositions pour la différentiation neuronale de cellules stromales pluripotentes
EP2440660A4 (fr) * 2009-06-10 2013-08-28 Brainstem Biotec Ltd Procédés, systèmes et compositions pour la différentiation neuronale de cellules stromales pluripotentes
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JP2013091611A (ja) * 2011-10-25 2013-05-16 Gifu Univ 間葉系細胞の分化調節剤およびこれを用いた医薬、並びに間葉系細胞への分化調節作用を有する物質のスクリーニング方法
JP2019509065A (ja) * 2016-03-16 2019-04-04 サイナータ セラピューティクス リミテッド コロニー形成培地及びその使用
JP7048977B2 (ja) 2016-03-16 2022-04-06 サイナータ セラピューティクス リミテッド コロニー形成培地及びその使用
JP2020516242A (ja) * 2017-04-12 2020-06-11 レバティス エスエーＲｅｖａｔｉｓ Ｓａ 哺乳動物筋由来幹細胞の新規用途
CN113621580A (zh) * 2021-08-18 2021-11-09 山东科金生物发展有限公司 一种增强脂肪间充质干细胞体外扩增活性的试剂

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