WO2010050328A1 - Inhibiteur de métastase de tumeur - Google Patents

Inhibiteur de métastase de tumeur Download PDF

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WO2010050328A1
WO2010050328A1 PCT/JP2009/066992 JP2009066992W WO2010050328A1 WO 2010050328 A1 WO2010050328 A1 WO 2010050328A1 JP 2009066992 W JP2009066992 W JP 2009066992W WO 2010050328 A1 WO2010050328 A1 WO 2010050328A1
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nucleotide sequence
mir143
nucleic acid
seq
mir145
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Japanese (ja)
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孝広 落谷
充彦 尾崎
松木　泰
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国立がんセンター総長が代表する日本国
大日本住友製薬株式会社
国立大学法人鳥取大学
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Publication of WO2010050328A1 publication Critical patent/WO2010050328A1/fr

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a drug that suppresses tumor metastasis, a medicine using the drug, a method for determining tumor metastasis risk, an agent for determining tumor metastasis risk, and a substance having an action of inhibiting tumor metastasis It is related with the screening method of this.
  • the present invention relates to a nucleic acid that suppresses metastasis of solid cancer, more specifically osteosarcoma.
  • miRNA is an endogenous non-coding RNA of about 20 to 25 bases encoded on the genome. miRNA is first transcribed from the miRNA gene on the genomic DNA as a primary transcript (Primary miRNA, hereinafter referred to as “Pri-miRNA”) with a length of several hundred to several thousand bases, and then processed. Pre-miRNA (precusor miRNA) having a hairpin structure of about 60 to 110 bases. After that, it moves from the nucleus into the cytoplasm and is further processed into a double-stranded mature miRNA of about 20-25 bases.
  • Primary miRNA Primary miRNA
  • Pre-miRNA precusor miRNA having a hairpin structure of about 60 to 110 bases. After that, it moves from the nucleus into the cytoplasm and is further processed into a double-stranded mature miRNA of about 20-25 bases.
  • RISC protein-binding protein
  • More than 1000 types of miRNA are known in humans and mice, and each of them has been suggested to regulate the expression of a plurality of target genes and participate in various life phenomena such as cell proliferation and differentiation.
  • miRNA involved in hematopoietic cell and neuronal cell differentiation for example, see Non-Patent Document 2.
  • miRNAs involved in cancer cell growth have been reported, and miRNA expression patterns can be used for clinical diagnosis of cancer, and miRNA expression can be suppressed to suppress cancer cell growth.
  • Patent Document 3 discloses the expression control pattern of microRNA 143 (hereinafter referred to as miR143) and pathway analysis of genes controlled by miR143, and Non-Patent Document 3 discloses that miR143 is expressed in colon cancer cells. In addition, as a result of experiments in which miR143 was introduced into colon cancer cells, miR143 has been shown to suppress cell growth of colon cancer.
  • Non-Patent Document 4 reports the results of examining the expression patterns of miR21, 31, 143 and 145 in tumor tissues of various stages of colon cancer (Colorectal) patients. It has been disclosed that miRNAs whose expression is increased in tumor tissues of colon cancer (Colorectal) patients are miR21 and 31, and miR143 and 145 have decreased expression.
  • tumor cells released from the primary lesion invade the vascular system, and when they reach a distant organ, they take a process of engrafting and starting to proliferate. Even with complete removal, cancerous cells can often metastasize patients to death.
  • metastasis to the lung causes direct or indirect death of the patient, that is, suppressing lung metastasis has been a major challenge in the treatment of osteosarcoma.
  • miRNAs involved in metastasis of tumors, particularly osteosarcoma have been no reports on miRNAs involved in metastasis of tumors, particularly osteosarcoma.
  • an object of the present invention is to provide a drug for controlling (suppressing) tumor, particularly osteosarcoma metastasis, a method for determining the risk of tumor metastasis, and a method for searching for a substance that can inhibit tumor metastasis. is there.
  • the present inventors obtained new knowledge about miRNA involved in osteosarcoma metastasis, and studied the tumor metastasis inhibitory effect of miR143 and miR145 using the knowledge. As a result, these miRNAs, particularly miR143 were excellent.
  • the present invention was completed by finding that it has an effect of suppressing tumor metastasis.
  • a tumor metastasis inhibitor comprising a nucleic acid comprising miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and having a function equivalent to miR143.
  • a tumor metastasis inhibitor according to [1] wherein the nucleic acid is single-stranded or double-stranded.
  • miR143 is a nucleotide having the nucleotide sequence represented by SEQ ID NO: 1.
  • nucleic acid is RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof, or a modified product thereof.
  • nucleic acid is RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a modified product thereof.
  • nucleic acid containing miR143 is at least one nucleic acid selected from the group consisting of miR143 and a precursor thereof.
  • precursor is pri-miRNA or pre-miRNA of miR143.
  • a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and represented by miR145 or SEQ ID NO: 3
  • a tumor metastasis inhibitor comprising a nucleotide sequence having 70% or more identity with a nucleotide sequence and a nucleic acid comprising a nucleotide having a function equivalent to miR145.
  • nucleic acid comprising miR143 or a nucleotide sequence represented by SEQ ID NO: 1 having a nucleotide sequence having 70% or more identity and having a function equivalent to miR143, or (b) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and represented by miR145 or SEQ ID NO: 3
  • An agent for preventing a disease caused by tumor metastasis comprising a nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% identity or more and having a function equivalent to miR145.
  • (A) (a) a nucleic acid comprising miR143 or a nucleotide sequence having a nucleotide sequence having 70% or more identity with SEQ ID NO: 1 and having a function equivalent to miR143, or (b) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and represented by miR145 or SEQ ID NO: 3
  • a nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity to the nucleotide sequence and having a function equivalent to miR145;
  • B A therapeutic agent for tumors that is used in combination with an antitumor agent.
  • An agent for determining the risk of tumor metastasis comprising a nucleic acid probe capable of specifically detecting miR143 or miR143 and miR145.
  • a method for searching for a substance that can inhibit tumor metastasis or tumor cell invasion ability comprising the following steps: (I) contacting a test substance with a cell capable of measuring the expression of miR143 or miR143 and miR145; (II) measuring the expression level of miR143 or miR143 and miR145 in a cell contacted with a test substance, and comparing the expression level with the expression level of miR143 or miR143 and miR145 in a control cell not contacted with the test substance; And (III) a test substance that increases the expression level of miR143 or miR143 and miR145 is selected as a substance that can inhibit tumor metastasis or tumor cell invasion ability based on the comparison result of (II) above. .
  • nucleic acid comprising a nucleotide sequence comprising miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and having a function equivalent to miR143 is administered to a mammal A method for suppressing tumor metastasis in the mammal.
  • a nucleic acid comprising an effective amount of miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and having a function equivalent to that of miR143 for mammals, and Metastasis of a tumor in the mammal comprising administering a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR145 or SEQ ID NO: 3 and having a function equivalent to miR145 How to suppress.
  • Effective amount for mammals (a) a nucleic acid comprising miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and comprising a nucleotide having a function equivalent to miR143, or (b) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and represented by miR145 or SEQ ID NO: 3
  • a method for preventing a disease caused by tumor metastasis in a mammal comprising administering a nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity with a nucleotide sequence and having a function equivalent to miR145.
  • [20] It consists of a nucleotide sequence having 70% or more identity with the effective amount of (A) (a) miR143 or the nucleotide sequence represented by SEQ ID NO: 1, and has a function equivalent to that of miR143.
  • a nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity with a nucleotide sequence and having a function equivalent to miR145 and (B) a method for treating a tumor in said mammal, comprising administering an antitumor agent .
  • a nucleic acid comprising miR143 or a nucleotide sequence having a nucleotide sequence having 70% or more identity with SEQ ID NO: 1 and having a function equivalent to miR143 for use in suppressing tumor metastasis.
  • nucleic acid comprising a nucleotide sequence comprising miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and having a function equivalent to miR143, for use in suppressing tumor metastasis; And a combination comprising a nucleic acid comprising miR145 or a nucleotide sequence represented by SEQ ID NO: 3 having a nucleotide sequence having 70% or more identity and having a function equivalent to miR145.
  • nucleic acid for use in the prevention of diseases caused by tumor metastasis, (a) a nucleic acid comprising miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and comprising a nucleotide having a function equivalent to miR143, or (b) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and represented by miR145 or SEQ ID NO: 3
  • a combination comprising a nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity to the nucleotide sequence and having a function equivalent to miR145.
  • [24] for use in treating a tumor (A) (a) a nucleic acid comprising miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and comprising a nucleotide having a function equivalent to miR143, or (b) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and represented by miR145 or SEQ ID NO: 3
  • a nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity to the nucleotide sequence and having a function equivalent to miR145 and (B) a combination comprising an antitumor agent.
  • a tumor metastasis inhibitor a method for determining tumor metastasis risk, an agent for determining tumor metastasis risk, and tumor metastasis inhibition, which show an inhibitory effect on tumor, particularly osteosarcoma metastasis
  • action which can be provided can be provided.
  • FIG. 1 is a diagram showing the results of miRNA microarray analysis in Reference Example 1.
  • FIG. 1 is originally a color photograph.
  • FIG. 2 is a scheme of a method for assaying the function of invasive capacity and proliferation of cells by introducing miRNA into 143B cells.
  • FIG. 3 shows the results of a 143B cell infiltration test in which miRNA was introduced.
  • FIG. 4 shows the results of a 143B cell proliferation test in which miRNA was introduced.
  • FIG. 5 shows the results of creating a mouse osteosarcoma metastasis model.
  • FIG. 5 is originally a color photograph.
  • FIG. 6 shows the administration protocol of miR143 to the mouse osteosarcoma metastasis model.
  • FIG. 7 shows lung metastases 4 weeks after administration of miR143 to a mouse osteosarcoma metastasis model.
  • FIG. 7 is originally a color photograph.
  • miR143 has a function of suppressing tumor invasion and metastasis (see Examples, described later), tumor (preferably solid cancer, more preferably osteosarcoma) It was found that it can be used as an active ingredient of a metastasis inhibitor.
  • the present invention An agent comprising a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and A nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by miR143 or SEQ ID NO: 1 and having a function equivalent to that of miR143, and a nucleotide sequence represented by miR145 or SEQ ID NO: 3 And a nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity and having a function equivalent to that of miR145.
  • the agent of the present invention is useful as a tumor metastasis inhibitor or an agent for treating or preventing a disease caused by tumor metastasis.
  • the nucleic acid of the present invention is RNA, RNA-DNA chimeric nucleic acid (hereinafter referred to as chimeric nucleic acid) or hybrid nucleic acid.
  • the chimera nucleic acid means a single-stranded or double-stranded nucleic acid containing RNA and DNA in a single nucleic acid
  • a hybrid nucleic acid is a double-stranded nucleic acid in which one strand is RNA or It refers to a nucleic acid in which the other strand is DNA or a chimeric nucleic acid.
  • the nucleic acid of the present invention is single-stranded or double-stranded.
  • Double-stranded embodiments include double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, RNA / chimeric nucleic acid hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid, and chimeric nucleic acid / DNA hybrid.
  • the nucleic acid of the present invention is preferably single-stranded RNA, single-stranded chimeric nucleic acid, double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, RNA / chimeric nucleic acid hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid, or chimeric nucleic acid / DNA hybrid, more preferably single-stranded RNA, single-stranded chimeric nucleic acid, double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid or RNA / chimeric nucleic acid hybrid .
  • the length of the nucleic acid of the present invention is not limited as long as it has an activity to inhibit the invasive ability of mammalian (preferably human) tumor cells.
  • the length of the nucleic acid of the present invention is, for example, about 200 bases or less, preferably about 130 bases or less, more preferably about 50 bases or less, and most preferably 30 bases or less. As a minimum, it is 15 bases or more, for example, Preferably, it is 17 bases or more.
  • the length of a nucleic acid in the case where a nucleic acid forms a double-stranded structure by taking a hairpin loop type structure is considered as a single-stranded length.
  • MiR143 is a molecule already known and typically refers to what is called mature miRNA.
  • miR143 includes isomers of miR143. Specifically, for example, it means a nucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 (UGAGAUGAAGCACUGUAGCUC) (MIMAT0000435).
  • the mature miR143 means single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1.
  • nucleic acid of the present invention When incorporated into a tumor cell, the nucleic acid of the present invention has an activity of inhibiting the invasive ability of the cell.
  • “nucleotide having a function equivalent to miR143” means a substance that forms a hybrid with miR143 target mRNA under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.). . More specifically, when miR143 target mRNA is hybridized under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.) and taken into tumor cells, the cells infiltrate. It means a nucleotide having an activity of inhibiting the ability.
  • Tumor cells are usually mammalian (eg, rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, monkey, human, preferably human) cells.
  • Tumor types include osteosarcoma, esophageal cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, colon cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, renal cancer, breast cancer , Bladder cancer, ovarian cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, Kaposi sarcoma, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, myoma, skin cancer, etc. And myeloma and leukemia.
  • the type of tumor is preferably solid cancer, more preferably osteosarcoma.
  • a nucleic acid has an activity of inhibiting the invasive ability of tumor cells can be confirmed by, for example, the following assay. That is, after culturing 143B cells, which are human osteosarcoma cell lines having metastatic potential, in 1 ⁇ 10 6 cells / 6 cm dish overnight, 30 nM of nucleic acid is introduced by DharmaFECT transfection (GE Healthcare Biosciences). The cells after 48 hours are subjected to cell invasion assay using CytoSelect TM 96-Well Cell Invasion Assay kit (Cell Biolab), and the number of infiltrated cells after 20 hours is counted.
  • 143B cells which are human osteosarcoma cell lines having metastatic potential
  • nucleotide sequence of “nucleotide having a function equivalent to miR143” used in the present invention is 70% or more, preferably 80% or more, more preferably 90% or more, and further preferably, the nucleotide sequence represented by SEQ ID NO: 1. It has 95% or more identity.
  • Identity refers to an optimal alignment when two nucleotide sequences are aligned using mathematical algorithms known in the art (preferably the algorithm uses one or the other of the sequences for optimal alignment). The percentage of identical nucleotide residues to all overlapping nucleotide residues) (which can be considered the introduction of gaps to both).
  • NCBI BLAST-2 National Center for Biotechnology Information Basic Local Alignment Search Tool
  • nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 one or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 have been deleted, substituted, inserted or added
  • a sequence for example, (1) a nucleotide sequence in which 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 have been deleted, (2) A nucleotide sequence obtained by adding 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides to the nucleotide sequence represented by SEQ ID NO: 1, and (3) a nucleotide represented by SEQ ID NO: 1.
  • nucleotide sequence in which 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides are inserted into the sequence (4) a nucleotide represented by SEQ ID NO: 1 A nucleotide sequence in which 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides in the tide sequence are replaced with other nucleotides, or (5) the above (1) to (4) A nucleotide sequence in which mutations are combined (in this case, the total number of nucleotides mutated is 1 to 6 (preferably 1 to 3, more preferably 1 or 2)).
  • the nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 is preferably 15 bases or more (preferably 17 bases or more, more preferably) contained in the nucleotide sequence represented by SEQ ID NO: 1.
  • the nucleic acid includes “miR143” or “nucleotide having a function equivalent to miR143”.
  • nucleic acids of the present invention may be modified to be resistant to various degrading enzymes.
  • the modified form of the present invention includes various modifications, including sequence modifications, within the range of nucleotides having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and having a function equivalent to miR143. Modified products are included.
  • modifications in the modified form include, for example, those in which the sugar chain part is modified (for example, 2′-O methylation), those in which the base part is modified, phosphate group or hydroxyl part is modified (For example, biotin, amino group, lower alkylamine group, acetyl group and the like), but not limited thereto.
  • the nucleic acid itself may be modified. Further, for example, it may be a synthetic nucleic acid comprising the same region as miR143 and a complementary region that is complementary to the sequence by 60% to less than 100%. It may be a synthetic RNA molecule as described in WO2006 / 627171.
  • the nucleic acid of the present invention may have an additional base at the 5 'or 3' end.
  • the length of the additional base is usually 5 bases or less.
  • the additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples include, but are not limited to, ', uuuuuu-3'.
  • nucleic acid of the present invention include nucleic acids such as mature miR143 and its precursor.
  • Another preferred embodiment of the nucleic acid of the present invention is to use a nucleic acid comprising a nucleotide having an activity similar to that of mature miRNA, such as miR143 mimic synthesized so as to mimic endogenous mature miR143. Can do.
  • Commercially available products can also be used.
  • Pre-miR TM miRNA precursor molecule manufactured by Ambion (by merger, Life Technologies, as of September 2009) can be exemplified.
  • the precursor of miR143 means a nucleic acid capable of producing mature miR143 in a cell as a result of intracellular processing or cleavage of a double-stranded nucleic acid.
  • Examples of the precursor include miR143 pri-miRNA and pre-miRNA.
  • the pri-miRNA is a primary transcript (single-stranded RNA) of the miRNA gene, and usually has a length of about several hundred to several thousand bases.
  • Pre-miRNA is a single-stranded RNA having a hairpin structure generated by pri-miRNA undergoing intracellular processing, and usually has a length of 90 to 110 bases.
  • pri-miRNA and pre-miRNA of miR143 are known molecules, and are disclosed in, for example, the miRBase database (http://microrna.sanger.ac.uk/) created by Sanger Laboratories. Has been.
  • a suitable pre-miRNA of miR143 a single-stranded RNA consisting of the nucleotide sequence represented by the following SEQ ID NO: 2 can be mentioned: GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUGAAGCACUGUAGCUCAGGAAGAGAGAAGUUGUUCUGCAGC (hsa-miR143 MI0000459)
  • a single-stranded nucleic acid in which a nucleotide sequence represented by SEQ ID NO: 1 (first sequence) and its complementary sequence (second sequence) are linked via a hairpin loop part A nucleic acid in which the first sequence has a shape of a double-stranded structure with the second sequence by taking a hairpin loop type structure is also a preferred embodiment of the nucleic acid of the present invention.
  • the nucleic acid of the present invention is obtained by isolating from a mammalian cell (human cell or the like) using a known method, or by chemically synthesizing, or by using a gene recombination technique. be able to. It is also possible to use commercially available nucleic acids as appropriate. miR143 mimic is available, for example, from Ambion (LifeologiesTechnologies as of September 2009).
  • the agent of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to an effective amount of the nucleic acid of the present invention, and is applied as a pharmaceutical in the form of a pharmaceutical composition.
  • Examples of pharmaceutically acceptable carriers include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, citric acid, Fragrances such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspensions such as methylcellulose and polyvinylpyrrolidone, dispersants such as surfactants, water, physiological Although diluents, such as salt solution, base wax, etc. are mentioned, it is not limited to them.
  • excipients such as sucrose and starch
  • binders such as cellulose and methylcellulose
  • disintegrants such as starch and carboxymethylcellulose
  • lubricants such as magnesium stearate and aerosil
  • citric acid Fragrances such as menthol
  • preservatives such as sodium benzoate and sodium bisulfite
  • stabilizers
  • the agent of the present invention can further contain a reagent for nucleic acid introduction.
  • the nucleic acid introduction reagent include atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly (ethyleneimine) (PEI) Cationic lipids such as can be used.
  • the nucleic acid of the present invention can be efficiently delivered to the target tumor cells and efficiently incorporated into the cells.
  • the agent of the present invention can be administered to mammals orally or parenterally, but it is desirable that the agent of the present invention be administered parenterally, usually parenterally. Administered.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • the preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials.
  • the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.
  • the content of the nucleic acid of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the whole pharmaceutical composition.
  • the dosage of the agent of the present invention varies depending on the purpose of administration, administration method, tumor type, size, and the situation of the subject of administration (sex, age, body weight, etc.).
  • the amount of the nucleic acid of the present invention is preferably 1 pmol / kg or more and 10 nmol / kg or less, and 2 nmol / kg or more and 50 nmol / kg or less for systemic administration. It is desirable to administer such dose 1 to 10 times, more preferably 5 to 10 times.
  • the agent of the present invention is a mammal (eg, rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, so that the nucleic acid of the present invention, which is an active ingredient thereof, is delivered to tumor tissue (tumor cells). It is safely administered to monkeys and humans.
  • the agent of the present invention is administered to a tumor patient, a patient after treatment of a tumor having a risk of tumor metastasis, or the like. Tumor metastasis can be suppressed, and diseases caused by tumor metastasis can be treated or prevented. Therefore, the agent of the present invention is useful as a tumor metastasis inhibitor or an agent for preventing a disease caused by tumor metastasis.
  • tumor suppression means that tumor cells reach a different site from the primary lesion and suppress the secondary generation of a tumor at the site.
  • tumors to which the agent of the present invention can be applied include osteosarcoma, esophageal cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, colon cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, Biliary tract cancer, renal cancer, breast cancer, bladder cancer, ovarian cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, Kaposi sarcoma, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, muscle tumor
  • solid cancers such as skin cancer and retinoblastoma, myeloma, leukemia, malignant lymphoma, myeloma, malignant melanoma, hemangioma, polycythemia vera, neuroblastoma and the like.
  • blood cell malignancies including breast cancer, lung cancer, renal cancer, multiple myeloma, thyroid cancer, prostate cancer, adenocarcinoma, leukemia and lymphoma; head and neck cancer; gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, liver cancer
  • malignant tumors of the female reproductive tract including ovarian cancer, endometrial cancer, and cervical cancer
  • bladder cancer brain tumors including neuroblastoma; sarcoma, osteosarcoma; and malignant melanoma or squamous epithelium
  • metastatic cancer such as skin cancer including cancer.
  • the agent of the present invention is preferably applicable to the above solid cancer, more preferably osteosarcoma.
  • diseases caused by tumor metastasis include metastatic cancer, respiratory failure due to tumor growth and cancerous pleurisy.
  • miR143 or a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 has the same function as miR143.
  • a nucleic acid containing nucleotides hereinafter referred to as the first nucleic acid
  • An agent comprising a combination of a nucleic acid containing hereinafter referred to as a second nucleic acid is provided.
  • the first nucleic acid is as described in the above section (1. Agent of the present invention), and the second nucleic acid is described in the section of (1. Agent of the present invention) except that miR143 is replaced with miR145. It is the same as the contents.
  • MiR145 is a nucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 (GUCCAGUUUUCCCAGGAAUCCCU) (MIMAT0000437), and a suitable precursor (pre-miRNA) of miR145 is represented by SEQ ID NO: 4 below.
  • a single-stranded RNA consisting of the following nucleotide sequence: CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGAUGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCUUGAGGUCAUGGUU (hsa-miR145 MI0000461)
  • the administration timing of the first nucleic acid and the second nucleic acid is not limited, and the first nucleic acid and the second nucleic acid are simultaneously administered to the administration subject. It may be administered or may be administered after a time difference.
  • the dosage of the first nucleic acid and the second nucleic acid is not particularly limited as long as the prevention / treatment of the applicable disease can be achieved, and administration is performed within the dosage range described in the above section (1. Agent of the present invention). Although it is possible to enhance the tumor metastasis inhibitory activity by the combined use, the first nucleic acid or the second nucleic acid can be used in an amount smaller than the dose described in the above section (1. Agent of the present invention). It is expected to exert the same effect as when administered alone.
  • the administration form of the first nucleic acid and the second nucleic acid is not particularly limited as long as the first nucleic acid and the second nucleic acid are combined at the time of administration.
  • Examples of such administration forms include (1) administration of a single preparation obtained by simultaneously formulating the first nucleic acid and the second nucleic acid, and (2) the first nucleic acid and the second nucleic acid.
  • An agent comprising a combination of the first nucleic acid and the second nucleic acid can be formulated by a conventional method according to the description in the above section (1. Agent of the present invention).
  • the preparation containing the first nucleic acid and the preparation containing the second nucleic acid may be administered at the same time.
  • the formulation containing the second nucleic acid may be administered first, followed by the formulation containing the first nucleic acid, or the formulation containing the first nucleic acid first, A formulation containing the second nucleic acid may be administered.
  • the time difference varies depending on the active ingredient to be administered, dosage form, and administration method.
  • the preparation containing the second nucleic acid examples include a method of administering a preparation containing the first nucleic acid within 1 minute to 3 days after administration, preferably within 10 minutes to 1 day, more preferably within 15 minutes to 1 hour.
  • a method of administering a preparation containing the first nucleic acid first after administration of the preparation containing the first nucleic acid, it is within 1 minute to 1 day, preferably within 10 minutes to 6 hours, more preferably from 15 minutes to 1
  • a method of administering a preparation containing the second nucleic acid within a time can be mentioned.
  • the present invention provides a tumor therapeutic agent comprising the nucleic acid of the present invention and an antitumor agent in combination.
  • the antitumor agent that can be used in the concomitant drug of the present invention is not particularly limited, but preferably has an activity of suppressing the growth of the tumor itself.
  • antitumor agents include not only microtubule agonists such as taxanes, but also antimetabolites, DNA alkylating agents, DNA binding agents (platinum preparations), anticancer antibiotics, and the like.
  • amrubicin hydrochloride irinotecan hydrochloride, ifosfamide, etopocydastet, gefitib, cyclophosphamide, cisplatin, trastuzumab, fluorouracil, mitomycin C, imatinib mesylate, methotrexate, rituxan, and adriamycin.
  • the administration timing of the nucleic acid of the present invention and the antitumor agent is not limited, and the nucleic acid of the present invention and the antitumor agent are administered simultaneously to the administration subject. Alternatively, administration may be performed with a time difference.
  • the dosage of the nucleic acid of the present invention is not particularly limited as long as it can achieve prevention / treatment of the applicable disease, and can be administered within the dosage range described in the above section (1. Agent of the present invention).
  • the dose of the antitumor agent can be determined according to the dose adopted when the antitumor agent is administered as a single agent in the clinic.
  • the administration mode of the nucleic acid of the present invention and the antitumor agent is not particularly limited as long as the nucleic acid of the present invention and the antitumor agent are combined at the time of administration.
  • Examples of such administration forms include (1) administration of a single preparation obtained by simultaneously formulating the nucleic acid of the present invention and an antitumor agent, and (2) separating the nucleic acid of the present invention and the antitumor agent separately.
  • An agent comprising a combination of the nucleic acid of the present invention and an antitumor agent can be formulated by a conventional method according to the description in the above section (1. Agent of the present invention).
  • the dosage form of the antitumor agent is selected according to the dosage form adopted when the antitumor agent is administered as a single agent in clinical practice. I can do it.
  • the nucleic acid-containing preparation of the present invention and the anti-tumor agent-containing preparation may be administered at the same time.
  • the preparation containing the antitumor agent may be administered first, followed by the preparation containing the nucleic acid of the present invention, or the preparation containing the nucleic acid of the present invention may be administered first, and then the antitumor agent May be administered.
  • the time difference varies depending on the active ingredient to be administered, dosage form, and administration method. For example, when a preparation containing an antitumor agent is administered first, a preparation containing an antitumor agent was administered.
  • Examples thereof include a method of administering a preparation containing the nucleic acid of the present invention within 1 minute to 3 days, preferably within 10 minutes to 1 day, more preferably within 15 minutes to 1 hour.
  • the preparation containing the nucleic acid of the present invention is administered first, after administration of the preparation containing the nucleic acid of the present invention, it is 1 minute to 1 day, preferably 10 minutes to 6 hours, more preferably 15 minutes to
  • the method of administering the formulation containing an antitumor agent within 1 hour is mentioned.
  • two or more kinds of antitumor agents may be used.
  • the present invention measures the expression level or concentration of miR143 or miR143 and miR145 in a tumor or peripheral blood, and a negative correlation between the expression level or the concentration and the incidence of metastasis
  • the present invention provides a method for determining the risk of metastasis of a tumor, including determining the risk of metastasis of a tumor.
  • the expression level or concentration of miR143 or miR143 and miR145 in a tumor or peripheral blood (preferably serum derived from peripheral blood) removed from a patient to be measured is measured.
  • tumor types to which the determination method of the present invention can be applied include tumors to which the agent of the present invention described in detail in the above section (1. Agent of the present invention) can be applied.
  • the determination method of the present invention is preferably applicable to the solid cancer, more preferably osteosarcoma.
  • the miR143 or miR145 whose expression level or concentration is measured in the determination method of the present invention includes mature type, pri-miRNA, and pre-miRNA.
  • the expression level and concentration of miR143 or miR145 can be measured by a method known per se using a nucleic acid probe that can specifically detect the miRNA.
  • the measuring method include RT-PCR, Northern blotting, in situ hybridization, nucleic acid array and the like.
  • it can be measured by a commercially available kit (for example, TaqMan (registered trademark) MicroRNA Cells-to-CT TM Kit).
  • the nucleic acid probe capable of specifically detecting miR143 or miR145 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably included in the nucleotide sequence represented by SEQ ID NO: 1 or 3.
  • the nucleic acid probe may contain an additional sequence (a nucleotide sequence that is not complementary to the polynucleotide to be detected) as long as specific detection is not hindered.
  • the nucleic acid probe may be an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase).
  • fluorescent substances eg, fluorescamine, fluorescein isothiocyanate, etc.
  • luminescent substances eg, luminol, luminol derivatives, luciferin, lucigenin, etc.
  • a quencher quenching substance
  • the fluorescence is detected by separating the fluorescent substance and the quencher during the detection reaction.
  • the nucleic acid probe may be any of DNA, RNA, and chimeric nucleic acid, and may be single-stranded or double-stranded.
  • the nucleic acid probe or primer can be synthesized according to a conventional method using an automatic DNA / RNA synthesizer based on the information of the nucleotide sequence represented by SEQ ID NO: 1 or 3, for example.
  • the risk of tumor metastasis is determined based on the measured expression level or concentration of miR143 or miR143 and miR145. As shown in Examples described later, the lower the expression level of miR143 in tumor tissue, the higher the incidence of distant metastasis. The determination is made based on such a negative correlation between the expression level or concentration of miR143 or miR143 and miR145 and the incidence of tumor metastasis.
  • a tumor is removed from a tumor patient without metastasis (negative control) and a patient with metastasis (positive control) (or peripheral blood is obtained (preferably serum thereof)), and the subject patient's miR143 or miR143 and miR145
  • the expression level (or concentration) of is compared to that of the positive and negative controls.
  • a correlation diagram between the expression level (or concentration) of miR143 or miR143 and miR145 in the tumor and the incidence of metastasis is prepared in advance, and miR143 or miR143 and miR145 in the tumor (or peripheral blood) removed from the subject patient.
  • the expression level may be compared with its correlation diagram.
  • the comparison of the expression level (or concentration) is preferably performed based on the presence or absence of a significant difference.
  • the risk of tumor metastasis is relatively high Can be determined. Conversely, when the expression level or concentration of miR143 or miR143 and miR145 to be measured is relatively high, it can be determined that the risk of tumor metastasis is relatively low.
  • the present invention also provides an agent (referred to as agent (II) of the present invention) for determining the risk of tumor metastasis, comprising a nucleic acid probe capable of specifically detecting the above-described miR143 or miR143 and miR145.
  • agent (II) of the present invention can be a kit for determining the risk of distant metastasis of a tumor or the life prognosis of a tumor patient. By using the agent (II) of the present invention, the risk of tumor metastasis can be easily determined by the above-described determination method.
  • the nucleic acid probe is usually in the form of an aqueous solution dissolved at an appropriate concentration in water or an appropriate buffer (eg, TE buffer, PBS, etc.), or the nucleic acid probe is immobilized on a solid support. In the embodiment of the nucleic acid array, it is included in the agent (II) of the present invention.
  • the agent (II) of the present invention may further contain other components necessary for carrying out the method as a constituent, depending on the measurement method of miR143 or miR143 and miR145.
  • the agent (II) of the present invention can further contain a blotting buffer, a labeling reagent, a blotting membrane, and the like.
  • the agent (II) of the present invention can further contain a labeling reagent, a chromogenic substrate, and the like.
  • a method for searching for a substance capable of inhibiting tumor metastasis or tumor cell invasion ability The present invention also includes assessing whether a test substance enhances the expression of miR143 or miR143 and miR145. Alternatively, a method for searching for a substance that can inhibit the invasion ability of tumor cells and a substance that can be obtained by the method are provided. In the search method of the present invention, a substance that up-regulates the expression of miR143 or miR143 and miR145 is selected as a substance that can inhibit tumor (particularly osteosarcoma) metastasis or tumor cell (particularly osteosarcoma cell) invasion ability.
  • test substance used in the search method of the present invention may be any known compound or novel compound, for example, using nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, combinatorial chemistry technique.
  • the prepared compound library, random peptide library, natural components derived from microorganisms, animals and plants, marine organisms, and the like can be mentioned.
  • the search method of the present invention includes the following steps: (I) contacting a test substance with a cell capable of measuring the expression of miR143 or miR143 and miR145; (II) measuring the expression level of miR143 or miR143 and miR145 in a cell contacted with a test substance, and comparing the expression level with the expression level of miR143 or miR143 and miR145 in a control cell not contacted with the test substance; And (III) a test substance that increases the expression level of miR143 or miR143 and miR145 is selected as a substance that can inhibit tumor metastasis or tumor cell invasion ability based on the comparison result of (II) above. .
  • miR143 or miR143 and miR145 whose expression levels are measured include mature forms, pri-miRNAs and pre-miRNAs, but preferably the sum or maturation of expression levels of all these types
  • the expression level of the type is measured, more preferably the expression level of the mature type.
  • a cell capable of measuring expression refers to a cell capable of evaluating the expression level of the miRNA to be measured. Examples of the cells include cells that can naturally express the miRNA to be measured.
  • the measurement target that is, a cell that can naturally express miR143 or miR143 and miR145 is not particularly limited as long as it can potentially express miR143 or miR143 and miR145, and the cell may be a mammal (eg, human, mouse, etc.).
  • the contact between the test substance and cells capable of measuring the expression of miR143 or miR143 and miR145 is performed in a culture medium.
  • the culture medium is appropriately selected depending on the cells capable of measuring the expression of miR143 or miR143 and miR145.
  • a minimal essential medium MEM containing about 5 to 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM).
  • the culture conditions are appropriately determined in the same manner.
  • the pH of the medium is about 6 to about 8
  • the culture temperature is usually about 30 to about 40 ° C.
  • the culture time is about 12 to about 72 hours.
  • the measurement of the expression level of miR143 or miR143 and miR145 can be performed according to the method described in the section of (3. Method for determining tumor metastasis risk).
  • the comparison of expression levels can be preferably performed based on the presence or absence of a significant difference.
  • the expression level of miR143 or miR143 and miR145 in the control cells not contacted with the test substance is the expression level measured in advance compared to the measurement of miR143 or miR143 and miR145 expression level in the cells contacted with the test substance. Even if it is, the expression level measured simultaneously may be sufficient, but it is preferable that it is the expression level measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.
  • the substance that up-regulates the expression level of miR143 or miR143 and miR145 obtained as a result of the comparison is selected as a substance that can inhibit tumor metastasis or tumor cell invasion ability.
  • the compound obtained by the search method of the present invention is useful as a candidate substance for the development of a new tumor metastasis inhibitor.
  • nucleotide sequence is described using the RNA sequence for the sake of convenience, but this does not mean that the nucleic acid identified by the SEQ ID NO represents only RNA, and U It should be understood that (uracil) is replaced with T (thymine) to indicate the nucleotide sequence of DNA or chimeric nucleic acid.
  • 143B cells form tumors when inoculated into mouse knee joints, and then form metastases in the lung, whereas HOS cells do not cause lung metastases.
  • the 143B cell is a cell line prepared by transforming HOS cells with the v-Ki-ras gene. That is, although both have high genetic background similarity, they exhibit completely different kinetics of metastasis, suggesting that miRNAs with different expression are involved in metastasis.
  • HOS cells and 143B cells were grown in DMEM medium supplemented with 10% horse serum and 10% fetal bovine serum (FBS), and RNA from both cell lines was mirVana RNA Isolation kit (Ambion Inc. (As of September, 2008, Life Technologies, Inc.)) was used for extraction, and miRNA microarrays (Agilent microarrays Ver. 1.0) were used to compare expression patterns. As a result, 9 types of miRNAs whose expression levels were more than doubled and 9 types of miRNAs of less than half were identified in 143B cells (highly metastatic strains) compared to HOS sputum cells (non-metastatic strains) (FIG. 1).
  • Test Example 1 Functional analysis of osteosarcoma metastasis-related candidate miRNA and identification of tumor metastasis-related candidate
  • a cell line 143B Fluc stably introduced by introducing the firefly luciferase gene pGL3control (Promega) into 143B cells was established. Functional analysis was performed.
  • MiRNAs miRNA-28, 99b, 133b, 140, 143, 145, 149, 193b, 335) whose expression was decreased in 143B cells are pre-miR TM miRNA precursor molecules (hereinafter referred to as “miRNA mimic” for convenience).
  • miRNA mimic pre-miR TM miRNA precursor molecules
  • miRNA 143B Fluc cells were grown in DMEM medium supplemented with 10% horse serum and 10% fetal bovine serum (FBS), cultured overnight in 1x10 6 cells / 6cm dish, then DharmaFECT transfection ( GE Healthcare Biosciences Co., Ltd.) introduced miRNA mimic (Ambion (manufactured by Life Technologies, as of September 2009 by merger)) according to the attached protocol.
  • FBS fetal bovine serum
  • Reference Example 2 Creation of a mouse osteosarcoma metastasis model Tumors (primary foci) are formed daily by inoculating 1.5x10 6 of the 143B Fluc prepared in Test Example 1 into the knee joints of nude mice, and can be detected by biological imaging in the early mouse after 2 weeks A mouse model of osteosarcoma spontaneous lung metastasis with pulmonary metastasis was constructed. In the primary lesion in the model, tumor cells increased histologically with the destruction of existing tissues including bone tissue, while in the lung, tumor cell masses similar to the tumor cells in the primary lesion were occasionally found. The formation of metastases was confirmed (FIG. 5).
  • Example 1 Effect of miR143 hyperfunction on osteosarcoma metastasis
  • Pre-miR TM miRNA143 precursor molecule (hereinafter referred to as “miRNA143 mimic” for convenience, using the mouse model of spontaneous pulmonary metastasis created in Reference Example 2) At that time, the suppression of lung metastasis by Life Technologies))) was verified.
  • miRNA143 mimic for convenience, using the mouse model of spontaneous pulmonary metastasis created in Reference Example 2
  • 143B Fluc 1.5 ⁇ 10 6 mice from the next day, 0.25 ⁇ g / ⁇ l miR143 mimic / 0.05% atelocollagen solution was administered to 200 ⁇ l per tail vein 7 times every 3 days.
  • miR-NC1 manufactured by Ambion (as of September 2009, Life Technologies) as a result of merger
  • mice Four weeks after 143B Fluc knee joint inoculation, the mice were sacrificed and the lungs and primary lesions were removed. Macroscopic observation of the lung revealed many yellowish white nodules in the miR-NC1 (Ambion (Merged, Life Technologies, as of September 2009)) administration group, but miRNA143 mimic (Ambion ( As a result of the merger, no obvious nodules were observed in the group treated with Life Technologies) as of September 2009, except for mice whose lung metastases were detected by in vivo imaging. On the other hand, the weight of the primary lesion was 3.67 ⁇ 0.59 g in the miR-NC1 (Ambion (from merger, Life Technologies) as of September 2009)) administration group, miRNA143 mimic (Ambion (in September 2009)).
  • Example 2 Preparation of Atelocollagen Formulation 1 ml of an aqueous solution containing 500 ⁇ g / ml miRNA143 mimic (Pre-miR TM miRNA143 precursor molecule: manufactured by Ambion (as of September 2009, Life Technologies) as a result of merger) and 0.1% (w 1 ml of atelocollagen solution of / w) was mixed to obtain 2 ml of 0.25 ⁇ g / ⁇ l miR-143 mimic / 0.05% atelocollagen preparation.
  • Pre-miR TM miRNA143 precursor molecule manufactured by Ambion (as of September 2009, Life Technologies) as a result of merger)
  • 0.1% w 1 ml of atelocollagen solution of / w
  • the osteosarcoma metastasis inhibitor of the present invention is useful for treatment and prevention of diseases caused by osteosarcoma metastasis.
  • a tumor metastasis risk can be determined, and an agent for determining a tumor metastasis risk and a method for screening a substance having an action of inhibiting tumor metastasis can be provided.

Abstract

L'invention porte sur : un inhibiteur de métastase de tumeur comprenant un acide nucléique ou des acides nucléiques tels que miR143, miR143 et miR145 ou similaire; un procédé de détermination du risque d'une métastase d'une tumeur, le procédé comprenant la mesure du taux d'expression de miR143 ou des taux d'expression de miR143 et mi145 dans la tumeur; un procédé de criblage d'une substance capable d'inhiber la métastase d'une tumeur, le procédé comprenant la sélection d'une substance capable de réguler de manière positive le taux d'expression de miR143 ou les taux d'expression de miR143 et miR145 en tant que substance capable d'inhiber la métastase de la tumeur; et autres.
PCT/JP2009/066992 2008-10-27 2009-09-29 Inhibiteur de métastase de tumeur WO2010050328A1 (fr)

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