JP2012171894A - Tumor reducing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a treating agent for tumor, especially an agent for treating breast cancer, osteosarcoma or prostatic cancer.SOLUTION: There is provided the treating agent for the tumor, especially the breast cancer, osteosarcoma or prostatic cancer, composed of a nucleotide sequence having ≥70% identity with a nucleotide sequence represented by sequence identification number 2 which is a complementary chain sequence of miR-625*, and containing a nucleic acid having an activity in inhibiting functions of the miR-625* or a vector allowing expression thereof.

Description

  The present invention relates to a drug that suppresses tumor growth, a drug using the drug, a method for determining a tumor, a tumor diagnostic agent, a screening method for a substance having an action of inhibiting tumor growth, and the like. Specifically, it relates to a therapeutic agent for osteosarcoma, prostate cancer and breast cancer.

Currently, RNA interference (RNAi) technology is frequently used in life science research, and its usefulness has been widely confirmed. RNAi refers to a phenomenon in which mRNA is degraded in a sequence-specific manner by double-stranded RNA, resulting in suppression of gene expression. Since it was reported in 2001 that small double-stranded RNA of 21 bases can mediate RNAi in mammalian cells (Non-patent Document 1), siRNA (small interferece RNA) is frequently used as a method for suppressing the expression of target genes. Has been. siRNA is expected to be applied to pharmaceuticals and various refractory diseases including cancer.
A microRNA (miRNA) is a short RNA molecule consisting of approximately 22 base pairs discovered in a nematode in 1993 and binds to a complementary sequence in the 3 'untranslated region of the target gene messenger RNA. It is known to suppress the translation and regulate various life phenomena. So far, it has been reported that there are 1048 types of microRNA in humans and 672 types in mice (http://www.mirbase.org/). Interestingly, there are a plurality of target genes for one microRNA, and they are often genes involved in the same physiological function and the same signal transduction pathway. Therefore, the expression variation of only one type of microRNA can cause a dynamic physiological action change. So far, the relationship between various diseases and abnormal expression of microRNA has been suggested, and in particular, there are many reports on the relationship between cancer onset and malignant transformation (Non-patent Document 2).
For example, miR-15a and miR-16-1 are reported to be present on chromosome 13q14, and the region is missing in approximately 70% of chronic lymphocytic leukemia patients. Furthermore, germline mutations of these microRNAs have also been found in patients with chronic lymphocytic leukemia, and mice with the same mutations have been shown to exhibit a similar phenotype as chronic lymphocytic leukemia (non- Patent Document 3). It is also known that the microRNA family called let-7 is missing from the genome, leading to constitutive activation of the RAS gene, a proto-oncogene, leading to carcinogenesis (Non-patent literature) 4).
miR-625 * is the so-called passenger strand of miR-625, and has been shown to be encoded on chromosome 14q23.3 in humans (http://www.mirbase.org/). Passenger strands were thought to be byproducts produced during the process of maturation of microRNA, but in recent years it has become clear that several types of passenger strands have microRNA activity such as translational inhibitory activity. (Non-Patent Documents 5, 6, and 7). Currently, it is believed that two different mature microRNAs are produced from one microRNA gene. Neither miR-625 nor miR-625 * has been reported for gene deletion, amplification, hyperfunction, etc. in cancer tissues, nor has it been reported to control cell growth.

Elbashir, SM et al. Nature 2001; 411: 494-8. Cancer and chemotherapy Vol. 37, No. 3, March 2010 Calin, GA et al. Proc Natl Acad Sci USA 2002; 99: 15524-9. Johnson, SM et al. Cell 2005; 120: 635-47. Lin, EA et al. J Biol Chem 2009; 284: 11326-35. Okamura, K et al. Nat Struct Mol Biol 2008; 15: 354-63. Seitz, H et al. Curr Biol 2008; 18: 147-51.

  An object of the present invention is to provide a drug that suppresses tumor growth, a drug using the drug, a method for determining a tumor, an agent for determining a tumor, and a screening method for a substance having an action of inhibiting tumor growth. It is to provide.

As a result of intensive studies on miR-625 *, the present inventors have found that expression of miR-625 * is increased in breast cancer cells as compared to normal cells, and breast cancers with high malignancy are benign breast cancers. The expression of miR-625 * is markedly increased compared to cells, and the addition of anti-miR-625 * to breast, osteosarcoma and prostate cancer cells suppresses the growth of cancer cells I found out.
Based on these findings, the present inventors have demonstrated that tumors that inhibit miR-625 * functions such as anti-miR-625 * and miR-625 * decoy RNA have an effect of suppressing the growth of tumors. The present invention was completed by finding it useful as a therapeutic agent.

That is, the present invention relates to the following.
[1] A therapeutic agent for tumors containing miR-625 * inhibitor as an active ingredient.
[2] The agent according to [1] above, wherein the miR-625 * inhibitor is the following (a) or (b):
(A) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the complementary strand sequence of miR-625 * and comprising a nucleotide that inhibits the function of miR-625 *;
(B) An expression vector capable of expressing the nucleic acid of (a) above.
[3] The agent according to [2] above, wherein the nucleic acid or vector is single-stranded or double-stranded.
[4] The agent according to [2] or [3] above, wherein the nucleic acid is RNA comprising the nucleotide sequence represented by SEQ ID NO: 2 or a partial sequence thereof, or a modified product thereof.
[5] The agent according to [2] or [3] above, wherein the nucleic acid is RNA consisting of a nucleotide sequence represented by SEQ ID NO: 2 or a modified product thereof.
[6] The agent according to [2] above, wherein the nucleic acid is a decoy RNA against anti-miR-625 * or miR-625 *.
[7] (A) the agent according to any one of [1] to [6] above,
(B) A therapeutic agent for tumors, which is used in combination with an antitumor agent.
[8] The agent according to any one of [1] to [7] above, wherein the tumor is a solid cancer.
[9] The agent according to [8] above, wherein the solid cancer is breast cancer, osteosarcoma or prostate cancer.
[10] Based on the measurement of the expression level or concentration of miR-625 * in the test sample and the positive correlation between the expression level or the concentration and the cancer, the presence or absence of cancer or cancer A method for determining cancer, comprising determining the grade of malignancy.
[11] The method described in [10] above, wherein the cancer is breast cancer, osteosarcoma or prostate cancer.
[12] A cancer diagnostic agent comprising a nucleic acid probe capable of specifically detecting miR-625 *.
[13] The agent according to [12] above, wherein the cancer is breast cancer, osteosarcoma or prostate cancer.
[14] A method for searching for a substance capable of suppressing tumor growth or tumor cell growth, comprising the following steps:
(1) contacting a test substance with a cell capable of measuring the expression of miR-625 *;
(2) measuring the expression level of miR-625 * in cells contacted with the test substance, and comparing the expression level with the expression level of miR-625 * in control cells not contacted with the test substance; 3) Select a test substance that down-regulates the expression level of miR-625 * as a substance capable of suppressing tumor growth or tumor cell growth based on the comparison result of (2) above.

According to the present invention, it is possible to provide a drug that suppresses tumor growth, and a drug that uses the drug, particularly a drug that is effective for breast cancer, osteosarcoma, and prostate cancer.
In addition, according to the present invention, a cancer determination method capable of determining not only the presence or absence of cancer but also the malignancy of cancer, a diagnostic agent for carrying out the determination method, tumor growth or tumor cell growth It is also possible to provide a screening method for a substance having an action of suppressing the above.

It is the figure which showed the expression level of miR-625 * of MDA-MB-231 cell, MCF-10A cell, and MCF-7 cell. It is the figure which showed the MDA-MB-231 cell growth inhibitory effect of anti-miR-625 *. It is the figure which showed the growth inhibitory effect of anti-miR-625 * of 143B cell (osteosarcoma cell; left) and PC-3M cell (prostate cancer cell; right). It is the figure which showed the growth inhibitory effect of the MDA-MB-231 cell, 143B cell, and PC-3M cell of the miR-625 * inhibitor vector.

  Hereinafter, the present invention will be described in detail.

1. Agents of the present invention The present inventors have found that anti-miR-625 * suppresses the growth of breast cancer cells, osteosarcoma cells and prostate cancer cells, and anti-miR-625 * and miR-625 * It was found that miR-625 * inhibitors that inhibit miR-625 * functions such as decoy RNA can be used as an active ingredient of tumor therapeutic agents.
That is, the present invention provides an agent containing a miR-625 * inhibitor.
The agent of the present invention is useful as a cancer therapeutic agent or cell growth inhibitor, and is particularly useful for breast cancer, osteosarcoma and prostate cancer.

  miR-625 * is an already known molecule, and typically refers to what is called mature miRNA. Here, miR-625 * includes isomers of miR-625 *. Specifically, for example, it means a nucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 1 (registered as accession No. MIMAT0004808 in miRBase). Mature miR-625 * means a single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1.

The miR-625 * inhibitor may be any substance that inhibits the function of miR-625 *, and its action mechanism is not limited.
“Inhibiting miR-625 * function” means inhibiting the growth of cancer cells that express miR-625 *, specifically breast cancer cells, osteosarcoma cells, or prostate cancer cells. It means to suppress the growth of. Whether or not to inhibit the function of miR-625 * can be confirmed by a known method such as the method described in Examples.

Specific examples of the miR-625 * inhibitor include the following nucleic acids (hereinafter referred to as “the nucleic acid of the present invention”).
(A) a nucleic acid comprising a nucleotide sequence having 70% or more identity with a complementary strand sequence of miR-625 * and comprising a nucleotide that inhibits the function of miR-625 *, or (b) the nucleic acid of (a) above A nucleic acid which is an expression vector capable of expressing

  In the present invention, the nucleic acid is RNA, a chimeric nucleic acid of RNA and DNA (hereinafter referred to as a chimeric nucleic acid) or a hybrid nucleic acid. Here, the chimera nucleic acid means a single-stranded or double-stranded nucleic acid containing RNA and DNA in one nucleic acid, and 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 a 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 a 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 .

(A) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the complementary strand sequence of miR-625 * and comprising a nucleotide that inhibits the function of miR-625 * The length of the nucleic acid of the present invention is a mammal As long as it has an activity of inhibiting the growth of (preferably human) tumor cells, there is no upper limit to its length. However, considering the ease of synthesis, antigenicity problems, etc., 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. The lower limit is, for example, 15 bases or more, preferably 17 bases or more. In addition, 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.

When incorporated into tumor cells, the nucleic acid of the present invention has the activity of inhibiting the growth of cancer cells, particularly breast cancer cells, osteosarcoma cells, and prostate cancer cells.
In the present invention, the complementary strand sequence of miR-625 * specifically means the nucleotide sequence represented by SEQ ID NO: 2.
“Nucleotide that inhibits miR-625 * function” means that miR-625 * is hybridized under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.) and tumor growth Means something to suppress. More specifically, cancer cells, particularly breast cancer, that form a hybrid with miR-625 * under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.) and express miR-625 * It means those that suppress cell growth of cells, osteosarcoma cells or prostate cancer cells.

  Tumor cells are usually mammalian (eg, rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, monkey, human, preferably human) cells. Tumor types include breast cancer including breast and ductal cancer, lung cancer, pancreatic cancer, prostate cancer, osteosarcoma, esophageal cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, Colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, renal cancer, bladder cancer, ovarian cancer, cervical cancer, thyroid cancer, testicular tumor, Kaposi sarcoma, maxilla Examples thereof include solid cancers such as cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, myoma, skin cancer, myeloma, leukemia and the like. Specifically, the tumor is preferably breast cancer, osteosarcoma and prostate cancer. Whether or not a nucleic acid has an activity of suppressing cancer cell growth can be confirmed by using a cancer cell line such as MDA-MB-231 cell, 143B cell, or PC-3M cell. I can do it.

  The nucleotide sequence of “nucleotide that inhibits the function of miR-625 *” used in the present invention is 70% or more, preferably 80%, of the nucleotide sequence represented by SEQ ID NO: 2 which is the complementary strand sequence of miR-625 *. As described above, it has an identity of 90% or more, more preferably 95% or more.

  “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).

  In the present specification, the identity in the nucleotide sequence is determined using, for example, the homology calculation algorithm NCBI BLAST-2 (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (gap open = 5 penalty; gap extension = 2) Penalty; x_dropoff = 50; Expected value = 10; Filtering = ON) can be calculated by aligning the two nucleotide sequences.

  As the nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, one or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 2 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: 2 have been deleted, (2) A nucleotide sequence in which 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides are added to the nucleotide sequence represented by SEQ ID NO: 2; (3) a nucleotide represented by SEQ ID NO: 2 An amino acid sequence in which 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides are inserted in the sequence; (4) 1 to 6 in the nucleotide sequence represented by SEQ ID NO: 2 ( Good Or a nucleotide sequence in which 1 to 3 nucleotides, more preferably 1 or 2 nucleotides are substituted with other nucleotides, or (5) a nucleotide sequence in which the mutations (1) to (4) above are combined (this The total number of mutated nucleotides 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: 2 is preferably 15 bases or more (preferably 17 bases or more, more preferably) contained in the nucleotide sequence represented by SEQ ID NO: 2. A partial sequence of preferably 19 bases or more, most preferably 20 bases) or a sequence containing it.

Since a natural nucleic acid is easily degraded by a nucleolytic enzyme present in a cell, the nucleic acid of the present invention may be modified so as to be resistant to various degrading enzymes. The modified form of the present invention includes a modification of the sequence within the range of nucleotides having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 and having the activity of inhibiting the function of miR-625 *. The modified form in which various modifications including the above are included. Examples of 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.
Further, the nucleic acid itself may be modified. Further, for example, it may be a synthetic nucleic acid containing the same region as the complementary strand of miR-625 * 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, ', uuuuu-3'.

Specific examples of the nucleic acid of the present invention include, for example, decoy RNA for anti-miR-625 * and miR-625 *.
As used herein, “anti-miR-625 *” is a form of miR-625 * microRNA activity-inhibiting molecule, and refers to a nucleic acid that is complementary to the miR-625 * sequence. Since the RISC complex containing microRNA cleaves RNA having a sequence that is completely complementary to the microRNA, a sequence that is not completely complementary to the target microRNA (for example, the 10th position from the 3 ′ end of the fully complementary sequence) By using a RISC complex containing the target microRNA (Experimental Medicine vol.28, No. 18 (2010) 2999-3004).
In general, anti-miR is a chemically modified single-stranded nucleic acid designed to specifically bind to endogenous microRNA and inhibit its function. Chemical modification is performed with the aim of conferring resistance of anti-miR to in vivo nuclease and improving the stability of the duplex formed with microRNA. 2'-O-methyl RNA, locked nucleic acid ( LNA), Peptide nucleic acid (PNA), etc. (Singh SK, Nielsen P, Koshkin A et al. LNA (locked nucleic acids): Synthesis and high-affinity nucleic acid recognition. Chem Commun 455, 1998).

  In this specification, “decoy RNA for miR-625 *” has a partially complementary sequence to miR-625 * and is less susceptible to cleavage and degradation even after base pairing with miR-625 * 2 RNA with the following structure. Decoy RNA is the same as anti-miR in that its function is inhibited by binding to the target microRNA, but its structure is a stem-loop RNA strand with a bubble structure, and one molecule of decoy RNA It differs from anti-miR in that it has multiple target microRNA binding sequences inside. A typical decoy RNA includes a stem-loop structure in which a bubble portion is formed by opposing microRNA binding sequences. By inserting a 1-5 base linker sequence between the microRNA binding sequence and the stem structure, the binding between the microRNA binding sequence and the target microRNA can be facilitated. In addition, as a microRNA binding sequence, a sequence that is not completely complementary to the target microRNA (for example, a sequence in which 4 bases are inserted between the 10th and 11th bases from the 3 ′ end of the completely complementary sequence, etc.) is used. By doing so, it is possible to avoid the cleavage of the decoy RNA by the RISC complex containing the target microRNA (Experimental Medicine vol.28, No.18 (2010) 2999-3004).

  The nucleic acid of the present invention can be obtained by chemically synthesizing using a conventionally known method, or by producing using a gene recombination technique.

(B) An expression vector capable of expressing the nucleic acid of (a) As another specific example of the present invention, a nucleotide sequence having 70% or more identity with the above-mentioned complementary sequence of (a) miR-625 * And a vector designed to express a “nucleic acid comprising a nucleotide that inhibits the function of miR-625 *”.
An “expression vector” in the present specification includes, for example, genetic information that can be replicated in a host cell, can be propagated autonomously, and can be easily isolated and purified from the host cell. And an expression vector having a functionable promoter and a detectable marker, into which the nucleic acid of the present invention is introduced so as to be placed under the control of the promoter.
Specifically, autonomous origins of replication derived from viruses such as plasmids such as pRC / RSV and pRC / CMV (manufactured by Invitrogen), bovine papillomavirus plasmid pBPV (manufactured by Amersham Bioscience), and EB virus plasmid pCEP4 (manufactured by Invitrogen) And vectors such as vaccinia virus and the like.

In the expression vector of the present specification, a promoter capable of functioning in a host cell is operably linked upstream of the nucleic acid of the present invention, and this is incorporated into a vector as described above, whereby the nucleic acid of the present invention is incorporated into the host. Expression vectors that can be expressed in cells can be constructed. Here, “to be operably linked” means that when the expression vector is introduced into a host cell, the nucleic acid of the present invention is transcribed and expressed under the control of a promoter in the host cell. It means that the promoter and the nucleic acid of the present invention are bound.
The promoter used here may be any promoter that can function in the cell into which the gene of the present invention is introduced. For example, SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Rous sarcoma virus promoter (RSV promoter), β Examples include an actin gene promoter or a pol II promoter such as a mouse papilloma virus (MMTV) promoter. In order to accurately transcribe a short RNA, a pol III promoter is generally used. Examples of polIII promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoter, and human valine-tRNA promoter. Further, a sequence in which 4 or more Ts are continuous is used as a transcription termination signal. When using a polIII promoter, the linker sequence described above for anti-miR-625 * and miR-625 * and the gap sequence for avoiding cleavage should be designed so that 4 or more Us do not continue. Should.
What is necessary is just to use suitably the restriction enzyme used in order to introduce | transduce the nucleic acid of this invention into an expression vector from Takara Shuzo etc. suitably. A commercially available vector containing such a promoter upstream of the multicloning site may be used.
In general, a DNA in which a promoter capable of functioning in a host cell and the nucleic acid of the present invention are operably linked is incorporated into a vector that can be used in the host cell and introduced into the host cell. . When using a vector that already has a promoter that can function in the host cell, the nucleic acid of the present invention is placed downstream of the promoter so that the promoter of the vector and the nucleic acid of the present invention are operably linked. Insert it. For example, the plasmids pRC / RSV, pRC / CMV, etc. described above have a cloning site downstream of a promoter that can function in animal cells, and the nucleic acid of the present invention is inserted into the cloning site and introduced into animal cells. Thus, the nucleic acid of the present invention can be expressed. If it is necessary to induce higher expression, a ribosome binding region may be linked upstream of the nucleic acid of the present invention. Examples of the ribosome-binding region used include those described in reports by Guarente, L. et al. (Cell 1980; 20: 543-53) and Taniguchi et al. (Genetics of Industrial Microorganisms, 1982, p202, Kodansha). .

Commercially available nucleic acids can also be used for the nucleic acid of the present invention. Anti-miR ^™ miRNA inhibitor (Applied Biosystems) is commercially available as anti-miR-625 *, and miArrest ^™ miRNA inhibitors (catalog No. HmiR-AN0733-SN-) is commercially available as decoy RNA against miR-625 *. Examples include 5, -10, -20 (synthetic oligos), HmiR-AN0733-AM01, 02, 03, 04 (expression vectors) (both manufactured by Genecopoeia).

  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 polyvinylpyrrolide, dispersants such as surfactants, water, Although diluents, such as physiological saline, base wax, etc. are mentioned, it is not limited to them.

  In order to promote introduction of the nucleic acid of the present invention into tumor cells, the agent of the present invention can further contain a reagent for nucleic acid introduction. Examples of the nucleic acid introduction reagent include atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly (ethyleneimine) (PEI) Cationic lipids such as can be used.

  By including the agent of the present invention in atelocollagen, 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 the agent of the present invention is preferably administered parenterally.

Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) 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. Alternatively, 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.
As another preparation suitable for parenteral administration, a spray etc. can be mentioned.

  The content of the agent 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 systemic administration is preferably 2 nmol / kg or more and 50 nmol / kg or less. It is desirable to administer such a 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.

  Examples of tumors to which the agent of the present invention can be applied include breast cancer, lung cancer, pancreatic cancer, prostate cancer, osteosarcoma, esophageal cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, Ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, renal cancer, bladder cancer, ovarian cancer, cervical cancer, thyroid cancer, testicular tumor, Kaposi sarcoma, maxillary cancer, tongue Cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, muscle tumor, skin cancer, retinoblastoma and other solid cancer, myeloma, leukemia, malignant lymphoma, myeloma, malignant melanoma, Examples include hemangioma, polycythemia vera, neuroblastoma and the like.

Specifically, cancer is preferable as the tumor to which the agent of the present invention can be applied.
Since the agent of the present invention has an activity of suppressing the growth of breast cancer cells, osteosarcoma cells and prostate cancer cells, the agent of the present invention is used for cancer patients, particularly breast cancer patients, osteosarcoma patients, prostate cancer patients, etc. Can be used to treat cancer diseases.
Therefore, the agent of the present invention is extremely useful as a therapeutic agent for tumors.
It is desirable to administer the agent of the present invention to a patient whose miR-625 * expression level is increased in the tumor tissue.

2. Combined use of the agent of the present invention and an antitumor agent The combined use of the nucleic acid of the present invention and an antitumor agent provides an effect of more effectively suppressing the growth of the tumor itself, so that the tumor is treated radically. be able to. Accordingly, the present invention provides a tumor therapeutic agent comprising the above-described nucleic acid of the present invention and an antitumor agent in combination.

  The antitumor agent that can be used in the combination agent of the present invention is not particularly limited, but those having an activity of suppressing the growth of the tumor itself are preferable. Examples of such 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. Specific examples include amrubicin hydrochloride, irinotecan hydrochloride, ifosfamide, etoposidrastat, gefitinib, cyclophosphamide, cisplatin, trastuzumab, fluorouracil, mitomycin C, imatinib mesylate, methotrexate, rituxan and adriamycin.

In the combined use of the nucleic acid of the present invention and the antitumor agent, 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.
Examples of administration subjects include breast cancer patients, osteosarcoma patients, and prostate cancer patients.
It is desirable to administer the agent of the present invention to patients whose miR-625 * expression level is increased in tumor tissues.

  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. Such dosage forms include, for example,
(1) administration of a single preparation obtained by simultaneously formulating the nucleic acid of the present invention and an antitumor agent,
(2) Simultaneous administration by the same administration route of two kinds of preparations obtained by separately formulating the nucleic acid of the present invention and the antitumor agent,
(3) Administration of two types of preparations obtained by separately formulating the nucleic acid of the present invention and an antitumor agent with a time difference in the same administration route,
(4) Simultaneous administration of two types of preparations obtained by separately formulating the nucleic acid of the present invention and the antitumor agent through different administration routes,
(5) Administration of two types of preparations obtained by separately formulating the nucleic acid of the present invention and the antitumor agent at different time intervals in different administration routes (for example, in the order of nucleic acid of the present invention → antitumor agent) Administration, or administration in the reverse order).

  An agent comprising the nucleic acid of the present invention and an antitumor agent in combination can be formulated by a conventional method according to the description in the above section (1. Agent of the present invention). When the nucleic acid of the present invention and the antitumor agent are formulated separately, 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.

  When the above-described nucleic acid and antitumor agent of the present invention are separately formulated and administered together, 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. When administered at a time difference, 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. A method of administering the preparation containing the nucleic acid of the present invention within 1 minute to 3 days later, preferably within 10 minutes to 1 day, more preferably within 15 minutes to 1 hour can be mentioned. When administering the preparation containing the nucleic acid of the present invention first, after administering the preparation containing the nucleic acid of the present invention, 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.

In the combination agent of the present invention, two or more kinds of antitumor agents may be used.
The concomitant drug of the present invention can be applied to tumors described in detail as “tumor to which the agent of the present invention can be applied” in the above section (1. Agent of the present invention). Specifically, the combination agent of the present invention is preferably applied to breast cancer, osteosarcoma and prostate cancer.

3. Method for determining cancer The present invention measures the expression level of miR-625 * in a test sample and determines whether it is cancer based on a positive correlation between the expression level and cancer. The method of determining cancer including this is provided. Since the expression level of miR-625 * is significantly different between benign and malignant breast cancer, the method of the present invention determines not only the presence of cancer but also the malignancy of the cancer. It is particularly useful for determining breast cancer.

A test sample is a cell or tissue collected from a measurement subject. The test sample can be collected from the measurement subject according to a known method.
The test sample is not particularly limited, but mammary gland, bone tissue, and prostate are preferable, and mammary gland cells, osteoblasts, osteoclasts, bone cells, and prostate cells are more preferable.
In the determination method of the present invention, the expression level of miR-625 * in the test sample is measured.

  MiR-625 * whose expression level is measured in the determination method of the present invention includes mature type, pri-miRNA, and pre-miRNA, but preferably the sum of expression levels of all these types or mature type The expression level, more preferably the mature expression level, is measured.

For example, the expression level of miR-625 * can be measured by a method known per se using a nucleic acid probe that can specifically detect the miRNA. Examples of the measuring method include RT-PCR, Northern blotting, in situ hybridization, nucleic acid array and the like. Alternatively, it can be measured by a commercially available kit (for example, TaqMan (registered trademark) MicroRNA Cells-to-CT ^™ Kit).

  The nucleic acid probe capable of specifically detecting miR-625 * 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. Mention may be made of polynucleotides comprising the full-length contiguous nucleotide sequence or its complementary sequence.

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, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent materials (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent materials (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.). Alternatively, a quencher (quenching substance) that absorbs fluorescence energy emitted by the fluorescent substance may be further bound in the vicinity of the fluorescent substance (eg, FAM, VIC, etc.). In such an embodiment, 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 2, for example.

  Next, based on the measured miR-625 * expression level, it is determined whether the test sample is cancerous or whether the cancer is malignant. As shown in Examples below, breast cancer cells have higher miR-625 * expression levels than normal cells. Highly malignant breast cancer cells have higher expression levels of miR-625 * than benign breast cancer cells. Therefore, the above determination is made based on a positive correlation between the expression level of miR-625 * and cancer, or a positive correlation between the expression level of miR-625 * and malignancy of cancer.

For example, cells or tissues are collected from healthy or non-cancer patients (negative control) and cancer patients (positive control), and the miR-625 * expression level of the target patient is compared with that of the positive and negative controls Is done. Alternatively, a correlation diagram between the expression level of miR-625 * in a specific cell or tissue and cancer may be prepared in advance, and the expression level of miR-625 * in a test sample collected from the subject patient You may compare. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.
If the expression level of miR-625 * to be measured is relatively high based on the comparison result of the expression level of miR-625 *, the test sample is relatively likely to be cancer, or It can be determined that the test sample is relatively likely to be malignant cancer. Conversely, when the expression level of miR-625 * to be measured is relatively low, it can be determined that the possibility that the test sample is cancer is relatively low.

  The subject of the agent of the present invention is preferably a cancer patient whose miR-625 * expression level is high in a test sample, and therefore the method of the present invention is useful for screening patients.

  The present invention also relates to an agent for determining the presence or absence of malignancy of cancer (hereinafter referred to as “the agent (II) of the present invention), comprising a nucleic acid probe capable of specifically detecting the above-described miR-625 *. ").). The agent (II) of the present invention can be a kit for determining the presence or absence of malignancy of cancer in a test sample. By using the agent (II) of the present invention, it is possible to easily determine whether the test sample has cancer or malignancy 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 according to the method for measuring miR-625 *. For example, when Northern blotting or a nucleic acid array is used for measurement, the agent (II) of the present invention can further contain a blotting buffer, a labeling reagent, a blotting membrane, and the like. When in situ hybridization is used for measurement, the agent (II) of the present invention can further contain a labeling reagent, a chromogenic substrate, and the like.

4). Method for Searching for a Substance that can Suppress Tumor Growth The present invention also searches for a substance that suppresses tumor cell growth, including evaluating whether a test substance suppresses the expression of miR-625 *. And a substance obtainable by the method. In the search method of the present invention, the substance that down-regulates the expression of miR-625 * is a drug that suppresses the growth of tumor cells (particularly breast cancer, osteosarcoma and prostate cancer), that is, a substance that can suppress tumor growth Selected as.

  The test substance used in the search method of the present invention may be any known compound and novel compound, for example, using nucleic acids, carbohydrates, lipids, proteins, peptides, organic low molecular weight compounds, combinatorial chemistry techniques. 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:
(1) contacting a test substance with a cell capable of measuring the expression of miR-625 *;
(2) measuring the expression level of miR-625 * in cells contacted with the test substance, and comparing the expression level with the expression level of miR-625 * in control cells not contacted with the test substance; 3) Select a test substance that down-regulates the expression level of miR-625 * as a substance that can inhibit tumor growth or tumor cell growth based on the comparison result of (2) above.

  In the search method of the present invention, miR-625 * whose expression level is measured includes mature type, pri-miRNA, and pre-miRNA, but preferably the sum of expression levels of all these types or mature type Expression level, and more preferably mature expression level.

  “A cell capable of measuring expression” refers to a cell capable of evaluating the expression level of miR-625 * to be measured. Examples of the cells include cells that can naturally express miR-625 * to be measured.

  The measurement target, that is, a cell capable of naturally expressing miR-625 * is not particularly limited as long as it can potentially express miR-625 *, and the cell may be a mammal (eg, human, mouse, etc.). Primary cultured cells, cell lines derived from the primary cultured cells, and the like can be used. Examples of cells that can naturally express miR-625 * include tumor cells that are described in detail as “tumors to which the agent of the present invention can be applied” in the above section (1. Agent of the present invention). it can. Breast cancer cells, osteosarcoma cells and prostate cancer cells are preferred, and malignant breast cancer cells are more preferred.

  Contact between the test substance and cells capable of measuring the expression of miR-625 * is performed in a culture medium. The culture medium is appropriately selected depending on the cells capable of measuring the expression of miR-625 *. For example, a minimal essential medium (MEM) containing about 5-20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM) ) Etc. The culture conditions are appropriately determined in the same manner. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is about 12 to about 72 hours.

  The expression level of miR-625 * can be measured according to the method described in the section (3. Cancer determination method).

  The comparison of expression levels can be preferably performed based on the presence or absence of a significant difference. It should be noted that the expression level of miR-625 * in the control cells not contacted with the test substance is the expression level measured in advance with respect to the measurement of the expression level of miR-625 * in the cells contacted with the test substance. Although the expression level measured simultaneously may be sufficient, the expression level measured simultaneously is preferable from the viewpoint of the accuracy and reproducibility of the experiment.

  The substance that down-regulates the expression level of miR-625 * obtained as a result of comparison is selected as a substance that can suppress the increase in cancer, particularly a substance that can inhibit the increase in breast cancer, osteosarcoma and prostate cancer. Is done.

  The compound obtained by the search method of the present invention is useful as a candidate substance for development of a therapeutic agent for a new tumor.

  In the sequence listing of the present specification, the 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 sequence number indicates only RNA, It should be understood that by appropriately replacing U (uracil) with T (thymine), the nucleotide sequence of DNA or chimeric nucleic acid is also indicated.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Reference Example 1 Comparison of the expression level of miR-625 * in MDA-MB-231 cells, MCF-10A cells and MCF-7 cells
MDA-MB-231 cells are a cell line isolated from human breast cancer, are used as a model for high-grade breast cancer that has lost estrogen sensitivity, and are known to metastasize to lymph nodes. On the other hand, MCF-7 cells are estrogen-sensitive breast cancer cells, have low malignancy and no metastatic potential. MCF-10A cells are mammary epithelium-derived cells and are known as normal cells without tumorigenicity (Soule HD et al. Cancer Research 50: 6075-6086, 1990).
MDA-MB-231 and MCF-7 cells are cultured using RPMI medium supplemented with 10% fetal bovine serum (FBS). MCF-10A cells are cultured with MEGM BulletKit (Takara Bio product code CC-3150). And cultured. RNA was extracted from both cell lines using miRNeasy Mini kit (Qiagen) according to the protocol attached to the kit. Next, reverse transcription reaction was performed using the obtained RNA as a template using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems), miR-625 * TaqMan MicroRNA Assay, and U6 TaqMan MicroRNA Assay (Applied Biosystems). RNA 1.7μL, 100mM dNTPs (with dTTP) 0.05μL, MultiScribe Reverse Transcriptase (50U / μL) 0.33μL, 10x Reverse Transcription Buffer 0.5μL, RNase Inhibitor (20U / μL) 0.063μL, Nuclease-free water 1.387μL Then, 1 μL of TaqMan MicroRNA Assay (5 ×) was mixed and incubated at 16 ° C. for 30 minutes, 42 ° C. for 30 minutes, and 85 ° C. for 5 minutes. Subsequently, 4.5 μL of the 4-fold dilution of the reaction solution, 0.5 μL of TaqMan MicroRNA Assay (20 ×), 5 μL of TaqMan 2 × Universal PCR Master Mix, No AmpErase UNGa (Applied Biosystems) are mixed, and 95 ° C. for 10 minutes. MDA-MB-231, MCF-10A by carrying out PCR reaction using a 7300 Real Time PCR System (manufactured by Applied Biosystems) by repeating 40 times of incubation cycle of 95 ° C for 15 seconds and 60 ° C for 1 minute. The expression levels of miR-625 * and U6 in MCF-7 cells were quantified. The expression level of miR-625 * in each cell corrected with the expression level of U6 is shown as a relative amount with the expression level in MCF-10A cells being 1 (FIG. 1). As a result, it was found that the expression level of miR-625 * in MCF-7 cells and MDA-MB-231 cells was significantly increased as compared with MCF-10A. In addition, the expression level of miR-625 * in MDA-MB-231 cells was significantly higher than that in MCF-7 cells.

Example 1 Anti-miR-625 * Inhibition of MDA-MB-231 Cell Growth Using anti-miR-625 * (manufactured by Applied Biosystems) at each concentration shown in FIG. 2, using a Dharmafect reagent kit (Dharmacon) According to the procedure attached to the reagent kit, the cells were introduced into MDA-MB-231 cells seeded at a concentration of 1 × 10 ⁶ cells / well (6-well plate). The next day, cells with anti-miR-625 * introduced were replated at a concentration of 1 × 10 ⁴ cells / well (96-well plate), and 2 days later, Tetracolor one for cell proliferation assay (Seikagaku Corporation) was used. Cell proliferation activity was measured. In addition, cells into which microRNA negative control 1 (Applied Biosystems) was introduced were used as control cells. As a result, it was found that Anti-miR-625 * inhibited the growth of MDA-MB-231 cells in a concentration-dependent manner (FIG. 2). This result suggests that miR-625 * has the cell growth promoting activity of MDA-MB-231.

Example 2 Anti-miR-625 * anti-miR-625 * (applied biosystems) at concentrations of 143B cells (osteosarcoma cells) and PC-3M cells (prostate cancer cells) ) Using Dharmafect reagent kit (Dharmacon) and introduced into 143B cells and PC-3M cells seeded at a concentration of 1 × 10 ⁶ cells / well (6-well plate) according to the procedure attached to the reagent kit did. The next day, cells with anti-miR-625 * introduced were replated at a concentration of 1 × 10 ⁴ cells / well (96-well plate), and 2 days later, Tetracolor one for cell proliferation assay (Seikagaku Corporation) was used. Cell proliferation activity was measured. In addition, cells into which microRNA negative control 1 (Applied Biosystems) was introduced were used as control cells. As a result, it was found that Anti-miR-625 * also suppressed cell proliferation of cancer cells other than breast cancer cells (FIG. 3).

Example 3 Cancer Cell Growth Inhibitory Capacity of miR-625 * Inhibitor Vector
2 μg of miArrest miR-625 * vector base inhibitor (Genecopoeia) using Lipofectamine LTX (Invitrogen) according to the procedure attached to the reagent kit, 1 × 10 ⁶ cells / well (6-well plate) The cells were introduced into MDA-MB-231 cells, 143B cells and PC-3M cells seeded at a concentration. On the next day, the cells were replated at a concentration of 1 × 10 ⁴ cells / well (96-well plate), and cell proliferation activity was measured 2 days later using Tetracolor one for cell proliferation assay (manufactured by Seikagaku Corporation). As control cells, cells into which miArrest vector base inhibitor (Genecopoeia) was introduced were used. FIG. 4 shows the inhibition rate of the proliferation activity of miArrest miR-625 * vector-based inhibitor-introduced cells relative to control cells. As a result, it was found that the cell growth of miArrest miR-625 * vector-based inhibitor-introduced cells was suppressed. This result also suggests that miR-625 * has cancer cell growth-promoting activity and that cancer cell growth can be suppressed by suppressing the function of miR-625 *.

  The therapeutic agent for tumors of the present invention is useful for therapeutic agents and prevention of tumors, particularly for the treatment and prevention of breast cancer, osteosarcoma and prostate cancer. In addition, the cancer determination method of the present invention can determine not only cancers such as breast cancer, osteosarcoma and prostate cancer, but also malignancy of cancer, and to determine cancer. And a method for screening a substance having an action of suppressing tumor growth or tumor cell growth.

Claims (14)

  A therapeutic agent for tumors containing miR-625 * inhibitor as an active ingredient. The agent according to claim 1, wherein the miR-625 * inhibitor is the following (a) or (b):
(A) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the complementary strand sequence of miR-625 * and comprising a nucleotide that inhibits the function of miR-625 *;
(B) An expression vector capable of expressing the nucleic acid of (a) above.   The agent according to claim 2, wherein the nucleic acid or vector is single-stranded or double-stranded.   The agent according to claim 2 or 3, wherein the nucleic acid is RNA consisting of a nucleotide sequence represented by SEQ ID NO: 2 or a partial sequence thereof, or a modified form thereof.   The agent according to claim 2 or 3, wherein the nucleic acid is RNA consisting of a nucleotide sequence represented by SEQ ID NO: 2 or a modified form thereof.   The agent according to claim 2, wherein the nucleic acid is a decoy RNA against anti-miR-625 * or miR-625 *. (A) the agent according to any one of claims 1 to 6;
(B) A therapeutic agent for tumors, which is used in combination with an antitumor agent.   The agent according to any one of claims 1 to 7, wherein the tumor is a solid cancer.   The agent according to claim 8, wherein the solid cancer is breast cancer, osteosarcoma or prostate cancer.   Based on measuring the expression level or concentration of miR-625 * in the test sample and positive correlation between the expression level or the concentration and the cancer, the presence or absence of cancer or the malignancy of the cancer A method for determining cancer, comprising determining.   11. The method of claim 10, wherein the cancer is breast cancer, osteosarcoma or prostate cancer.   A cancer diagnostic agent comprising a nucleic acid probe capable of specifically detecting miR-625 *.   The agent according to claim 12, wherein the cancer is breast cancer, osteosarcoma or prostate cancer. A method for searching for a substance capable of suppressing tumor growth or tumor cell growth, comprising the following steps:
(1) contacting a test substance with a cell capable of measuring the expression of miR-625 *;
(2) measuring the expression level of miR-625 * in cells contacted with the test substance, and comparing the expression level with the expression level of miR-625 * in control cells not contacted with the test substance; 3) Select a test substance that down-regulates the expression level of miR-625 * as a substance capable of suppressing tumor growth or tumor cell growth based on the comparison result of (2) above.