WO2012121178A1

WO2012121178A1 - Tumor angiogenesis inhibitor

Info

Publication number: WO2012121178A1
Application number: PCT/JP2012/055476
Authority: WO
Inventors: 孝広落谷; 展慶小坂; 晴久井口
Original assignee: 独立行政法人国立がん研究センター; 大日本住友製薬株式会社
Priority date: 2011-03-04
Filing date: 2012-03-02
Publication date: 2012-09-13
Also published as: JPWO2012121178A1

Abstract

An inhibitor cited in (a) below, or a tumor therapeutic agent containing a nucleic acid cited in (b) below: (a) an miR-151-3p inhibitor, an miR-886-3p inhibitor, or an SMPD3 inhibitor; and (b) (1) a nucleic acid comprising a nucleotide sequence having at least 70% identity with a nucleotide sequence represented by any of SEQ ID NO: 1 to 6, or miR-29b-1*, has-miR-221*, miR-138, miR-584, miR-30a, or miR-146a, the nucleic acid containing a nucleotide having inhibitory activity on the expression of a target gene; or (2) a nucleic acid that is an expression vector capable of expressing the nucleic acid of (1).

Description

Tumor angiogenesis inhibitor

The present invention relates to a drug that suppresses tumor angiogenesis, a drug using the drug, a method for determining malignant cancer, an agent for determining malignant cancer, and a substance having an action of inhibiting tumor angiogenesis It is related with the screening method of this.

In recent years, hypotheses have been proposed that cancer cells are familiar with surrounding cells so as to be convenient for their own proliferation, invasion, and metastasis, and form a so-called “cancer microenvironment” (Non-patent Documents 1 and 2). The importance of intercellular signal transmitters derived from cancer cells has been suggested.
Tumor blood vessels are blood vessels that develop disorderly and have structural imperfections such as unequal caliber and insufficiency, not only failing to achieve the original purpose of blood vessels such as oxygen and nutrient supply to tissues. The cancer microenvironment is maintained at low oxygen, and cancer cells present therein acquire high drug resistance and invasion / metastasis ability (Non-patent Document 3). However, the molecular mechanism has not been elucidated, and a sufficient treatment for correcting tumor blood vessels has not been obtained.
Recent angiogenesis studies have shown that there is a mechanism to moderately suppress vascular endothelial growth factor (VEGF) signaling in normally formed blood vessels to prevent excessive angiogenesis (non-patented) References 17, 18). As one of the mechanisms, it has been suggested that the DLL4-Notch signal pathway suppresses the VEGF signal as a so-called negative feedback signal (Non-patent Document 19). It is known that angiogenesis abnormalities are caused when DLL4-Notch signal is inhibited.
DLL4 (Delta-like ligand 4) is a single-transmembrane type membrane protein that also functions as a ligand for the single-transmembrane Notch protein. Notch protein is cleaved by ADAM metalloproteinase and γ-secretase by DLL4 stimulation, and intracellular domain Notch intracellular domain (NICD) is produced. Next, it is known that NICD moves into the nucleus and activates the RBPJ / CSL transcription factor to control various gene expressions (Non-patent Document 20). Moreover, it is known that the expression of DLL4 is increased by VEGF, and it has been reported that the activation is mediated by Akt3 (Non-patent Document 21).

Sphingomyelin phosphodiesterase (SMPD) 3 is a membrane-bound enzyme having two transmembrane domains, and its physiological functions are diverse such as intracellular signal transduction and oxidative stress response. Although the primary sequence has little homology with bacterial SMPD as a whole, the limited amino acid residues of the enzyme activity center are conserved. As a role of SMPD3 in vivo, it has been clarified from studies of genetically-mutated mice that it is involved in bone formation and tooth formation (Non-patent Document 4). Regarding the relationship between cancer and SMPD3, only some methylation of the SMPD3 gene has been reported in some breast cancer cells (Non-Patent Document 5), and no report suggesting its function has been made.

MicroRNAs are known to play a very important role in cancer development, and so far many oncogenic microRNAs such as miR-16, miR-21, let-7 have been identified. Yes. In addition, miR-126 and miR-210 have been identified as microRNAs that control angiogenesis (Non-Patent Document 6), but the relationship between microRNAs and angiogenesis shown below is not known.

Although miR-29b-1 is known to be negatively regulated by c-Myc, Hedgehog, and NF-KappaB in bile duct cancer (Non-patent Document 7), miR-29b is a passenger sequence. There is no report on the expression fluctuation or physiological function of -1 * in cancer cells.
Although miR-221 * is known to exist in vivo as a passenger sequence of miR-221, it has not been reported to have a physiological effect as a microRNA.
It has been suggested by Srivastava et al. that miR-138 plays an important role in the myocardial development stage (Non-patent Document 8). In relation to cancer, it has been shown to suppress metastasis of Head and Neck squamous cell carcinoma (HNSCC cells) (Non-patent Document 9), but its effect on angiogenesis is not known.
miR-584 has a suppressed expression level in RCC cells, which are renal cancer cells, compared to HK-2 cells, which are normal kidney cells, and the ROCK-1 gene, which is a factor that promotes cell proliferation, Since it is a target gene, it is reported to be one of tumor suppressor genes in renal cancer (Non-patent Document 10). In addition, a report that the expression level is suppressed by Nasopharyngeal carcinoma-associated gene 6 (NGX6), one of the tumor suppressor genes, suggests that it suppresses tumor growth in cancer cells. (Non-Patent Document 11).
Although miR-30a is reported to have increased expression in a cancer cell line called A549, a type of cancer cell, cell proliferation ability even when miR-30a is transduced into normal cancer cells No changes were observed in tumorigenicity, drug sensitivity, etc. (Non-patent Document 12). Moreover, although it has been reported that the expression is increased in other cancer types such as esophageal cancer, the physiological action has not been shown yet (Non-patent Document 13).
miR-146a is known to be highly expressed not only in cancers such as papilloma thyroid cancer but also in autoimmune diseases such as rheumatism and psoriasis. It has been found that the most important target gene is a transcription factor called NF-κB having immune activation and cell growth promoting activity (Non-patent Document 14).
miR-151-3p has been reported to be upregulated in liver cancer, and miR-151-5p present on the same gene has been shown to have an effect of promoting metastasis (Non-patent literature) 15) However, there has been no report on the function of miR-151-3p.
miR-886-3p has a binding sequence on the 3'UTR sequence of Stromal Derived Factor 1 (SDF1, CXCL12), a cell chemotactic factor, and is known to negatively regulate its expression in human stromal cells. It has been. In fact, transduction of miR-886-3p into HS27a cells suppresses Jurkat cell migration, and conversely, introduction of anti-miR-886-3p promotes Jurkat cell migration. (Non-Patent Document 16).
In recent years, it is becoming clear that microRNA is secreted from cells and functions as a signal transduction substance between cells (Non-patent Document 22). It has also been reported that secretory microRNAs are secreted in large quantities from cancer cells (Non-patent Document 23), so that secretory microRNAs derived from cancer cells are used to form cancer microenvironments such as tumor angiogenesis. Expected to be involved. However, a method for suppressing the function of these cancer cell-derived secreted microRNAs has not been established.
On the other hand, as a method for suppressing the activity of microRNA in the cell, an anti-miR method using a nucleic acid having a complementary sequence of the microRNA (anti-miR), a nucleic acid having a complementary sequence of a target mRNA of the microRNA (target -mask), a target-mask method that inhibits the binding of the microRNA to its target mRNA, and a vector (sponge vector or decoy vector) that expresses multiple 5 ′ end partial sequences (seed sequences) of the microRNA. A sponge method or a decoy method for trapping RNA is known (Non-patent Document 24).
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. (Non-patent Document 25). Here, the LNA modified nucleic acid refers to a nucleic acid derivative having two circular structures in which the 2′-position oxygen atom and the 4′-position carbon atom of the nucleic acid are bridged via a methylene residue. The locked nucleic acid (LNA) is also called 2 ', 4'-BNA (2'-O, 4'-C-methylene bridged nucleic acid). In non-clinical studies, it has been reported that LNA-modified anti-miR has been successfully systemically administered to non-human primates to successfully and effectively silence target microRNAs (Non-patents) Reference 26).
Natural nucleic acids (DNA and RNA) have a greater degree of freedom in form (conformation freedom) due to their chemical structure. For this reason, DNA-DNA and RNA-RNA duplex formation (hybridization) is thermodynamically disadvantageous, and improving binding affinity (hybridization ability) is a problem for nucleic acid drugs. . LNA increases the binding affinity for target DNA and RNA by thermodynamically stabilizing the 2 'oxygen atom and the 4' carbon atom via a methylene bridge, and It is known that nuclease (nucleolytic enzyme) resistance can be imparted (Non-patent Document 27).
However, specifically, it is not clear which nucleic acid in anti-miR can be maximally brought out by applying LNA modification (Non-patent Documents 28 and 29).

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

As a result of intensive studies, the present inventors have found that 24 types of microRNAs (miR-29b-1 *, miR-221 *, miR-138, miR- 584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR-140-3p, miR-593, miR-483-3p) and identified these microRNAs When miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a are introduced into cells, angiogenesis is suppressed, miR-151-3p or miR-886-3p It was found that angiogenesis is promoted when introduced into cells.
Furthermore, the present inventors have found that when breast cancer cells in which the SMPD3 gene is knocked down are transplanted into nude mice, angiogenesis in the tumor is suppressed and metastatic potential is also suppressed.
Based on these findings, the present inventors have confirmed that SMR3 inhibitors such as miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, and SMPD3 siRNA are tumors. It is useful as a therapeutic agent, and miR-151-3p inhibitors such as anti-miR-151-3p and miR-886-3p inhibitors such as anti-miR-886-3p are thought to inhibit angiogenesis. Therefore, the present invention was completed by finding that it is useful as a tumor therapeutic agent.

That is, the present invention relates to the following.
[1] The following (a) inhibitor or tumor therapeutic agent containing the nucleic acid according to (b):
(A) an miR-151-3p inhibitor, an miR-886-3p inhibitor, or an SMPD3 inhibitor,
(B) (1) a nucleotide sequence represented by any of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, or SEQ ID NOs: 1-6 A nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity and having a target gene expression suppressing activity, or (2) a nucleic acid that is a vector capable of expressing the nucleic acid of (1) above.
[2] The agent according to [1], wherein the miR-151-3p inhibitor is the nucleic acid according to the following (1) or (2):
(1) a nucleic acid comprising a nucleotide sequence comprising 70% or more of the nucleotide sequence represented by SEQ ID NO: 9, and comprising a nucleotide having an activity of suppressing the function of miR-151-3p,
(2) A nucleic acid that is an expression vector capable of expressing the nucleic acid of (1) above.
[3] The agent according to [1], wherein the miR-886-3p inhibitor is the nucleic acid according to (1) or (2) below:
(1) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and comprising a nucleotide having an activity of suppressing the function of miR-886-3p,
(2) A nucleic acid that is an expression vector capable of expressing the nucleic acid of (1) above.
[4] The agent according to [1], wherein the SMPD3 inhibitor is a substance selected from the group consisting of the following (1) to (5):
(1) an antisense nucleic acid against a transcription product of a gene encoding SMPD3,
(2) a ribozyme nucleic acid for the transcription product of the gene encoding SMPD3,
(3) a nucleic acid having RNAi activity for a transcription product of a gene encoding SMPD3 or a precursor thereof,
(4) an antibody that binds to SMPD3,
(5) A low molecular weight compound that binds to SMPD3.
[5] The agent according to any one of [1] to [4], wherein the nucleic acid is single-stranded or double-stranded.
[6] The nucleic acid is any one of [1] to [3], wherein the nucleic acid is RNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6, 9, and 10 or a partial sequence thereof, or a modified form thereof. Agent.
[7] The nucleic acid according to any one of [1] to [3] and [6], wherein the nucleic acid is RNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6, 9, and 10, or a modified form thereof. Agent.
[8] The agent according to [2], wherein the nucleic acid is a single-stranded RNA comprising the nucleotide sequence represented by SEQ ID NO: 9 or SEQ ID NO: 54 or a modified product thereof.
[9] The agent according to [8], wherein the nucleic acid is a single-stranded RNA containing at least one or more modified nucleotides, either 2′-OMe modified nucleotides or LNA modified nucleotides.
[10] The agent according to [9], wherein the single-stranded RNA contains 3 or more LNA-modified nucleotides.
[11] The agent according to [9], wherein the single-stranded RNA contains 4 or more LNA-modified nucleotides.
[12] The agent according to [9], wherein the single-stranded RNA comprises 3 to 12 LNA modified nucleotides.
[13] The agent according to [9], wherein the single-stranded RNA comprises 4 to 12 LNA modified nucleotides.
[14] The agent according to [8] to [13], wherein the single-stranded RNA contains at least one LNA-modified nucleotide at both the 5 ′ end and the 3 ′ end.
[15] The agent according to [8] to [14], wherein all nucleotides are single-stranded RNAs consisting of modified nucleotides of either 2′-OMe modified nucleotides or LNA modified nucleotides.
[16] The agent according to [10], wherein the single-stranded RNA is a single-stranded RNA consisting of a nucleotide sequence represented by any one selected from SEQ ID NOs: 30 to 38 and 43 to 50.
[17] The agent according to [10], wherein the single-stranded RNA is a single-stranded RNA consisting of a nucleotide sequence represented by any one selected from SEQ ID NOs: 43 to 48 and 50.
[18] The agent according to any one of [1] to [17], which inhibits angiogenesis.
[19] The agent according to any one of [1] to [17] for inhibiting tumor angiogenesis.
[20] The agent according to any one of [1] to [17] for suppressing tumor metastasis.
[21] The agent according to any one of [1] to [20], wherein the tumor is breast cancer.
[22] The inhibitor or nucleic acid according to any one of [3] and [8] to [17] for suppressing angiogenesis abnormality due to overexpression of miR-151-3p.
[23] The inhibitor or nucleic acid according to any one of [1] to [17] for treating a tumor.
[24] The inhibitor or nucleic acid according to any one of [1] to [17] for inhibiting angiogenesis.
[25] The inhibitor or nucleic acid according to any one of [1] to [17] for inhibiting tumor angiogenesis.
[26] The inhibitor or nucleic acid according to any one of [1] to [17] for suppressing tumor metastasis.
[27] Use of the inhibitor or nucleic acid according to any one of [1] to [17] for producing a tumor therapeutic agent.
[28] Use of the inhibitor or nucleic acid according to any one of [1] to [17] for producing an angiogenesis inhibitor.
[29] Use of the inhibitor or nucleic acid according to any one of [1] to [17] for producing a tumor angiogenesis inhibitor.
[30] Use of the inhibitor or nucleic acid according to any one of [1] to [17] for producing a tumor metastasis inhibitor.
[31] A method for treating a tumor in a human, comprising administering the inhibitor or nucleic acid according to any one of [1] to [17] to the human.
[32] A method for inhibiting angiogenesis in a human, comprising administering the inhibitor or nucleic acid according to any one of [1] to [17] to the human.
[33] A method for inhibiting tumor angiogenesis in a human, comprising administering the inhibitor or nucleic acid according to any one of [1] to [17] to the human.
[34] A method for suppressing tumor metastasis in a human, comprising administering the inhibitor or nucleic acid according to any one of [1] to [17] to the human.
[35] miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100 in the test sample , MiR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR -140-3p, miR-593 or miR-483-3p is measured and the incidence of malignant cancer based on a positive correlation between the secreted level or concentration and malignant cancer A method for determining cancer, comprising determining the presence or absence of cancer.
[36] The method of [35], wherein the cancer is breast cancer.
[37] miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221 , MiR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR-140-3p An agent for determining malignant cancer, comprising a nucleic acid probe capable of specifically detecting miR-593 or miR-483-3p.
[38] The agent according to [37], wherein the cancer is breast cancer.
[39] A method for searching for a substance capable of suppressing tumor angiogenesis, comprising the following steps:
(1) contacting a test substance with a cell capable of measuring secretion or expression of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a;
(2) measuring the amount of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a secreted or expressed in cells contacted with the test substance, Comparing the amount of secretion or expression with the amount of secretion or expression in control cells not contacted with the test substance; and (3) based on the comparison result of (2) above, miR-29b-1 *, miR-221 * Select a test substance that up-regulates the secretion or expression level of miR-138, miR-584, miR-30a, or miR-146a as a substance that can suppress tumor angiogenesis.
[40] A method for searching for a substance capable of suppressing tumor angiogenesis, comprising the following steps:
(1) contacting a test substance with a cell capable of measuring the secretion, expression or function of miR-151-3p, miR-886-3p or SMPD3;
(2) Measure the secretion amount, expression level or function of miR-151-3p, miR-886-3p or SMPD3 in cells contacted with the test substance, and determine the secretion amount, expression level or function of the test substance Comparing with the secreted amount, expression level or function in the non-contacted control cells; and (3) secreted amount, expression of miR-151-3p, miR-886-3p or SMPD3 based on the comparison result of (2) above A test substance that down-regulates the amount or function is selected as a substance capable of suppressing tumor angiogenesis.
[41] consisting of the nucleotide sequence represented by SEQ ID NO: 9 or SEQ ID NO: 54, wherein all nucleotides consist of modified nucleotides of either 2′-OMe modified nucleotides or LNA modified nucleotides, and 3 to 12 LNAs Single-stranded RNA comprising modified nucleotides.
[42] The single-stranded RNA according to [41], wherein the single-stranded RNA comprises 4 to 12 LNA-modified nucleotides.
[43] The single-stranded RNA according to [41] or [42], wherein the single-stranded RNA contains at least one LNA-modified nucleotide at both the 5 ′ end and the 3 ′ end.
[44] The single-stranded RNA according to [42], comprising a nucleotide sequence represented by any one selected from SEQ ID NOs: 30 to 38 and 43 to 50.

According to the present invention, it is possible to provide an agent that suppresses tumor angiogenesis, in particular, a tumor therapeutic agent effective for breast cancer, and a medicine using the agent.
Further, according to the present invention, there are also a cancer determination method capable of determining the presence or absence of malignant cancer, an agent for carrying out the determination method, and a screening method for a substance having an action of suppressing tumor angiogenesis. Can be provided.

It is the figure which showed the metastasis | transition suppression of the breast cancer cell in a subcutaneous transplant model by SMPD3 knockdown. FIG. 5 shows that SMPD3 knockdown does not suppress breast cancer cell metastasis in the tail vein administration model. It is the figure which showed the evaluation result of intracellular miR-151-3p activity inhibitory activity of Anti- mmu-miR-151-3p. It is the figure which showed the result of stability evaluation in serum of Anti- mmu-miR-151-3p. It is the figure which showed the evaluation result of the double strand formation ability of Anti- mmu-miR-151-3p and mature type miR-151-3p. It is the figure which showed the evaluation result of the secretion-type mmu-miR-151-3p activity inhibitory activity of Anti- mmu-miR-151-3p. It is the figure which showed the evaluation result of the lymph node metastasis in the subcutaneous transplantation model of miR-151-3p hypersecretion cell. It is the figure which showed the evaluation result of tumor angiogenesis in the subcutaneous transplantation model of miR-151-3p hypersecretory cell. It is the figure which showed suppression of DLL4 signal by miR-151-3p. It is the figure which showed that miR-151-3p suppresses the luciferase activity of HEK293 cell which introduce | transduced hAkt3 | 3'UTR vector. It is the figure which showed that endogenous hAkt3 protein amount decreased by introduction of hsa-miR-151-3p. It is the figure which showed that the expression increase of DLL4 was canceled by knockdown of Akt3 gene.

Hereinafter, the present invention will be described in detail.

1. Agents of the present inventionThe inventors have miR-151 such as miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, anti-miR-151-3p It has been found that miR-886-3p inhibitors such as -3p inhibitors, anti-miR-886-3p, and SMPD3 inhibitors inhibit tumor angiogenesis and are useful as excellent tumor therapeutic agents.
That is, the present invention
(A) miR-151-3p inhibitor, miR-886-3p inhibitor, or SMPD3 inhibitor, and (b) (1) miR-29b-1 *, miR-221 *, miR-138, miR-584 MiR-30a, miR-146a, or a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 1 to 6 and having a target gene expression-suppressing activity, Or (2) a nucleic acid that is an expression vector capable of expressing the nucleic acid of (1) above,
Is to provide.
The agent of the present invention is useful as a therapeutic agent for tumors, and particularly useful for breast cancer. Since the agent of the present invention has angiogenesis inhibitory activity, it is expected to suppress tumor metastasis.

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 the hybrid nucleic acid is a double-stranded nucleic acid having one strand of RNA or DNA. 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 .

The length of the nucleic acid of the present invention is not limited as long as it has an activity to suppress angiogenesis in mammals (preferably humans). 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. That is, the length of the nucleic acid of the present invention is preferably 15 to 50 bases, more preferably 15 to 30 bases, and further preferably 17 to 30 bases. In addition, the length of a nucleic acid when a nucleic acid forms a double stranded structure by taking a hairpin loop type structure shall be considered as the length of a single strand.

The nucleic acid of the present invention has an activity of inhibiting angiogenesis when taken into cells, and particularly has an activity of inhibiting angiogenesis of tumor cells when taken into tumor cells.

The tumor cells are usually mammalian cells (for example, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys, humans, preferably humans). 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. Whether or not the nucleic acid has the activity of suppressing cell angiogenesis should be confirmed, for example, by using normal human umbilical vein endothelial cells (HUVEC cells) or normal human skin microvascular endothelial cells (HMVEC cells). I can do it.

(A) Nucleotide sequence represented by miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, or any one of SEQ ID NOs: 1 to 6 and 70% or more The nucleic acid miR-29b-1 *, which has a nucleotide sequence having the same identity and contains nucleotides that suppress target gene expression , is already a known molecule and is typically called a mature miRNA Is mentioned. Here, miR-29b-1 * includes microRNAs (isomers) having the same sequence as miR-29b-1 * and present at different positions on the genome. Specifically, for example, it means a nucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 1 (registered as accession No. MIMAT0004514 in miRBase). The mature miR-29b-1 * means a single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1.

MiR-221 * is an already known molecule and typically means what is called mature miRNA. Here, miR-221 * includes microRNAs (isomers) having the same sequence as miR-221 * and present at different positions on the genome. Specifically, for example, it means a nucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 2 (registered in miRBase as Accession No. MIMAT0004568). Mature miR-221 * means single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 2.

MiR-138 is an already known molecule, and typically refers to what is called mature miRNA. Here, miR-138 includes microRNAs (isomers) having the same sequence as miR-138 and existing at different positions on the genome. Specifically, for example, it means a nucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 3 (registered as accession No. MIMAT0000430 in miRBase). Mature miR-138 means single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 3.

MiR-584 is a known molecule and typically means what is called mature miRNA. Here, miR-584 includes microRNAs (isomers) having the same sequence as miR-584 and present at different positions on the genome. Specifically, for example, it means a nucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 4 (registered as AccessionBaseNo. MIMAT0003249 in miRBase). The mature miR-584 means a single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 4.

MiR-30a is an already known molecule, and typically means what is called mature miRNA. Here, miR-30a includes microRNAs (isomers) having the same sequence as miR-30a and existing at different positions on the genome. Specifically, for example, it means a nucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 5 (registered in miRBase as Accession No. MIMAT0000087). The mature miR-30a means a single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 5.

MiR-146a is a known molecule, and typically refers to what is called mature miRNA. Here, miR-146a includes microRNAs (isomers) having the same sequence as miR-146a and existing at different positions on the genome. Specifically, for example, it means a nucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 6 (registered as Accession No. MIMAT0000449 in miRBase). Mature miR-146a means a single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 6.

In the present invention, a nucleic acid comprising a nucleotide consisting of a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 1 to 6 is a nucleotide represented by any of SEQ ID NOs: 1 to 6 There is no limitation as long as the mRNA has a target gene expression suppressing activity having a nucleotide sequence complementary to the nucleotide sequence of the miRNA comprising the sequence. The nucleic acid preferably has an activity of suppressing angiogenesis.
In the present invention, “target gene expression inhibitory activity” refers to an activity of suppressing the expression of a target gene having a nucleotide sequence complementary to the target miRNA in mRNA. As an example, in the case of a nucleic acid comprising a nucleotide consisting of a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, the nucleotide represented by SEQ ID NO: 1 has the target gene expression suppressing activity. It has the activity which suppresses the expression of the target gene which has a nucleotide sequence complementary to a sequence in mRNA. The activity is, for example, by introducing into a cell both an expression vector having a synthetic sequence complementary to the target miRNA on the 3 ′ end side of the reporter gene (eg, luciferase) and a vector expressing the target miRNA. The effect of the expression on the expression of the target gene can be confirmed by measuring the activity of a reporter protein (for example, luciferase) (see the method described in Example 6 or 9), but is not limited thereto Instead, any method may be used as long as it is a known method used for evaluating the target gene expression inhibitory activity.
In the present invention, “having an activity to suppress angiogenesis” specifically means miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a. It means a nucleotide having an activity of forming a hybrid under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.) and suppressing angiogenesis. More specifically, a hybrid is formed with a nucleic acid comprising the nucleotide sequence represented by any one of SEQ ID NOS: 1 to 6 under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.), and When taken into a tumor cell, it means a nucleotide having an activity of suppressing angiogenesis of the cell. Whether or not the angiogenesis of cells is suppressed can be evaluated by a known method such as the method described in Examples.

The nucleotide sequence of “nucleotide having activity to suppress angiogenesis” used in the present invention is the sequence of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a It has 70% or more, preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more identity with the nucleotide sequence represented by any of Nos. 1-6.

“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 ratio of the same nucleotide residue to the total overlapping nucleotide residues in the case of introducing gaps into both).

In this 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 any one of SEQ ID NOs: 1 to 6, one or more nucleotides are missing from the nucleotide sequence represented by any of SEQ ID NOs: 1 to 6. Nucleotide sequences deleted, substituted, inserted or added, for example, (1) 1 to 6 (preferably 1 to 3, more preferably 1 or 1 in the nucleotide sequence represented by any of SEQ ID NOs: 1 to 6) (2) nucleotide sequence from which nucleotides are deleted, (2) 1 to 6 nucleotides represented by any one of SEQ ID NOs: 1 to 6 (preferably 1 to 3, more preferably 1 or 2) (3) 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides in the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6 Insert (4) 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides in the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6 are other nucleotides. Or (5) a nucleotide sequence in which the mutations of (1) to (4) above are combined (in this case, the sum of the 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 any of SEQ ID NOs: 1 to 6 is preferably a contiguous 15 nucleotide sequence contained in the nucleotide sequence represented by any of SEQ ID NOs: 1 to 6. A partial sequence of at least bases (preferably at least 17 bases, more preferably at least 19 bases, most preferably at least 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 product of the present invention has 70% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 1 to 6, and has the above-mentioned target gene expression inhibitory activity or angiogenesis inhibitory activity In the range of nucleotides having a, a modified product having various modifications including a modification of the sequence is included. Examples of modifications in the modified form include, for example, those in which the sugar chain moiety is modified (for example, 2'-O methylation, LNA), those in which the base moiety is modified, phosphate moieties or hydroxyl moieties Examples thereof include, but are not limited to, modified ones (for example, biotin, amino group, lower alkylamine group, acetyl group).
In addition, the 5 ′ end and 3 ′ end of the nucleotide chain may be modified with an amino group, polyethylene glycol, cholesterol, or the like.

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'.

Preferred embodiments of the nucleic acid of the present invention include nucleic acids such as mature miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a, and precursors thereof. Can do. Another preferred embodiment of the nucleic acid of the present invention is a nucleic acid comprising a nucleotide having the same activity as that of the mature miRNA, such as endogenous mature miR-29b-1 *, miR-221 *, miR-138, miR Nucleic acids that are synthesized so as to mimic -584, miR-30a, or miR-146a and retain these target gene expression-suppressing activities can be used. Commercially available products can also be used. For example, Pre-miR ^™ miRNA precursor molecule (Applied systems) can be exemplified.

miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a precursor is the intracellular processing or cleavage of double-stranded nucleic acids as a result of intracellular processing. Means a nucleic acid capable of giving rise to mature forms miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a. Examples of the precursor include pri-miRNA and pre-miRNA of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a. A pri-miRNA is a primary transcript (single-stranded RNA) of a 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.
miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a pri-miRNA and pre-miRNA are well-known molecules. MiRBase database: http://microrna.sanger.ac.uk/ etc.

Further, for example, a single strand in which the nucleotide sequence (first sequence) represented by any one of SEQ ID NOS: 1 to 6 and its complementary sequence (second sequence) are linked via a hairpin loop portion A nucleic acid which has a hairpin loop type structure, so that the first sequence has a double-stranded structure with the second sequence 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.

(B) Expression vector capable of expressing nucleic acid of (A) As another specific example of the present invention, the above-mentioned “miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a” , MiR-146a, or a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 1 to 6 and having a target gene expression inhibitory activity " Vectors designed for can be mentioned.
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 the nucleic acid of the present invention is transcribed and expressed in the host cell under the control of a promoter when the expression vector is introduced into 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 nucleic acid of the present invention is introduced. For example, SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Rous sarcoma virus promoter (RSV promoter), β Pol II promoters such as actin gene promoter, CMV early enhancer / chicken beta actin (CAG) promoter, SRα promoter, Thymidine Kinase (TK) promoter, elongation factor-1 α promoter, or mouse papilloma virus (MMTV) promoter However, it is common to use a polIII promoter in order to perform transcription of short RNA accurately. Examples of the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, and the like. Further, a sequence in which 4 or more Ts are continuous is used as a transcription termination signal.
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 multiple cloning 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 aforementioned plasmids pRC / RSV, pRC / CMV, etc. 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 further high 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 expression vectors for expressing the nucleic acid of the present invention can also be used. For example, as a commercial product of an expression vector for each of the precursors miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a and miR-146a, Precursor miRNA clones catalog No. HmiR0120-MR01 , 04 (miR-29b-1 *), HmiR0369-MR01, 04 (miR-221 *), HmiR0431-MR01, 04 (miR-138), HmiR0083-MR01, 04 (miR-584), HmiR0096-MR01, 04 (MiR-30a) and HmiR0087-MR01, 04 (miR-146a) (both manufactured by Genecopoeia).

(C) The miR-151-3p inhibitor miR-151-3p is a known molecule, and representative examples include what are called mature miRNAs. Here, miR-151-3p includes microRNA (miR-151-3p isomer) having the same sequence as miR-151-3p and present at different positions on the genome. Specifically, for example, human miR-151-3p (hsa-miR-151-3p) consisting of a nucleotide sequence represented by SEQ ID NO: 7 (registered as accession No. MIMAT0000757 in miRBase) can be mentioned. That is, mature miR-151-3p includes single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 7. Moreover, as a specific example of miR-151-3p, in addition to human miR-151-3p consisting of the nucleotide sequence represented by SEQ ID NO: 7 above, SEQ ID NO: 53 (registered in miRBase as Accession No. MIMAT0000161) And orthologs of other animal species such as mouse miR-151-3p (mmu-miR-151-3p) having the nucleotide sequence represented by

The miR-151-3p inhibitor may be any substance that inhibits the function of miR-151-3p, and its action mechanism is not limited.
As "function of miR-151-3p",
(1) Activity to suppress expression of a target gene having a nucleotide sequence complementary to miR-151-3p in mRNA (hereinafter, miR-151-3p target gene expression suppression activity), or (2) Cell angiogenesis Promoting activity,
Is mentioned.
The target gene expression suppression activity of miR-151-3p is, for example, an expression vector having a synthetic sequence complementary to miR-151-3p on the 3 ′ end side of a reporter gene (for example, luciferase) and miR-151- A vector expressing 3p can be introduced into a cell, and the effect of miR-151-3p expression on target gene expression can be confirmed by measuring the activity of a reporter protein (eg, luciferase). (See the method described in Example 6 or 9). In addition, the activity of promoting cell angiogenesis is achieved by, for example, introducing a vector expressing miR-151-3p into vascular endothelial cells capable of tube formation (for example, human umbilical vein endothelial cells HUVEC) on the extracellular matrix. Can be confirmed by measuring the degree of tube formation (see the method described in Example 10), but is not limited thereto, and is a known one used to evaluate angiogenic ability. Any method may be used as long as it is a method.
“Inhibiting the function of miR-151-3p” means having the activity of suppressing the above-mentioned function of miR-151-3p, and whether or not to inhibit the function of miR-151-3p. It can be confirmed by a known method such as the method described in Examples.

Specific examples of miR-151-3p inhibitors include the following nucleic acids.
(A) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the complementary strand sequence of miR-151-3p, and comprising a nucleotide that inhibits the function of miR-151-3p, or (b) the above (a) A nucleic acid that is an expression vector that expresses.
In the present invention, the complementary strand of miR-151-3p specifically refers to a nucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 9.

“Nucleotide inhibiting the function of miR-151-3p” means a hybrid with miR-151-3p under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.), and miR- Means what suppresses the function of 151-3p. More specifically, when miR-151-3p is hybridized under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.) and taken up into tumor cells, miR in the cells -151-3p means a nucleotide having the activity of suppressing the function of 3p.

The nucleotide sequence of “nucleotide having the activity of suppressing the function of miR-151-3p” used in the present invention is 70% or more of the nucleotide sequence represented by SEQ ID NO: 9 which is a complementary strand of miR-151-3p. , Preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more. Specific examples include nucleotides consisting of the nucleotide sequence represented by SEQ ID NO: 54.

As a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 9, one or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 9 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: 9 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: 9, and (3) a nucleotide represented by SEQ ID NO: 9 A nucleotide 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: 9 A nucleotide sequence in which nucleotides (preferably 1 to 3, more preferably 1 or 2) are substituted with other nucleotides, or (5) a nucleotide sequence in which the mutations (1) to (4) above are combined ( In this case, 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: 9 is preferably 15 or more consecutive nucleotides (preferably 17 or more nucleotides or more, preferably included in the nucleotide sequence represented by SEQ ID NO: 9). A partial sequence of preferably 19 bases or more, most preferably 21 bases) or a sequence containing it.

Specific examples of the nucleic acid of the present invention include, for example, decoy RNA for anti-miR-151-3p and miR-151-3p.
As used herein, “anti-miR-151-3p” is a form of miR-151-3p microRNA activity-inhibiting molecule and refers to a nucleic acid that is complementary to the miR-151-3p sequence.
In general, anti-miR is a chemically modified single-stranded nucleic acid designed to specifically bind to endogenous microRNA and inhibit its function. Therefore, the anti-miR-151-3p of the present invention may be modified so as to be resistant to various degrading enzymes. The modified product of the present invention includes a nucleotide sequence having the identity of 70% or more with the nucleotide sequence represented by SEQ ID NO: 9 and having the activity of suppressing the function of miR-151-3p. Modifications including various modifications including modifications are included.
For example, the phosphate residue (phosphate) of each nucleotide constituting the nucleic acid can be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, and the like. In addition, the 2′-position hydroxyl group of each sugar (ribose) is changed to OR (R═CH ₃ (2′-O-Me), F (2′-F), CH ₂ CH ₂ OCH ₃ (2′- O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.

The conformation of the sugar part of nucleic acids is dominated by C2'-endo (S-type) and C3'-endo (N-type). In single-stranded nucleic acids, they exist as an equilibrium between them, but double-stranded Is fixed to the N type. Therefore, in order to confer strong binding ability to the target RNA, LNA (Imanishi, which fixed the conformation of the sugar moiety to the N-type by cross-linking the 2 ′ oxygen and the 4 ′ carbon via a methylene residue. T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004; Singh SK, Nielsen P, Koshkin A, et al. LNA (locked nucleic acid acids): Synthesis and high-affinity nucleic acid recognition. Chem Commun. 455, 1998) and 2 'oxygen and 4' carbon were cross-linked via ethylene residues to fix the sugar conformation to N-type. ENA (Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003) can also be preferably used.
In addition, the 5 ′ end and 3 ′ end of the nucleotide chain may be modified with an amino group, polyethylene glycol, cholesterol, or the like.
Furthermore, Peptide nucleic acid (PNA), which is a nucleic acid synthesized with a peptide chain as a skeleton, can be used in place of phosphate sugar ester (Singh SK, Nielsen P, Koshkin A, et al. LNA (locked nucleic acid). acids): Synthesis and high-affinity nucleic acid recognition. Chem Commun. 455, 1998).

For example, the anti-miR-151-3p of the present invention comprises a modified nucleotide, preferably a 2′-O methyl modified nucleotide or an LNA modified nucleotide at any position of a single stranded nucleic acid (preferably a single stranded RNA). It may also contain two or more different modified nucleotides. Here, the LNA-modified nucleotide represents a modified nucleotide in which the 2′-position oxygen atom and the 4′-position carbon atom are cross-linked via a methylene group in RNA. A preferred form is a nucleic acid containing 3 or more (more preferably 4 or more), more preferably 3 to 12 (more preferably 4 to 12) LNA-modified nucleotides of single-stranded RNA. When the strand RNA is a single-stranded RNA containing 3 or more LNA-modified nucleotides, it is preferably a nucleic acid containing at least one LNA-modified nucleotide at both the 5 ′ end and the 3 ′ end. In addition, when the single-stranded RNA contains LNA-modified nucleotides, it is preferable that all remaining nucleotides that are not LNA-modified are 2′-O methyl-modified.
Specific examples of anti-miR-151-3p include a modified nucleic acid consisting of a nucleotide sequence represented by SEQ ID NOs: 43 to 50 (preferably SEQ ID NOs: 43 to 48 and 50) described in Example 10, and Examples And a modified nucleic acid having the nucleotide sequence represented by SEQ ID NOs: 30 to 38 described in FIG.

The anti-miR-151-3p of the present invention determines the target sequence of mRNA or initial transcript based on the sequence of miR-151-3p, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman). Etc.) can be prepared by synthesizing a complementary sequence thereto. In addition, anti-miR-151-3p containing the various modifications described above can be chemically synthesized by any known method.

Without being bound by any theory, anti-miR-151-3p is a cell or non-human animal (eg rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, monkey, human, preferably human) In animals (eg, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys, etc.) by forming a double strand with the target microRNA (ie, miR-151-3p), the cells or It is considered that the amount of the target microRNA that can exert a function of promoting angiogenesis in a non-human animal (ie, an effective amount) is decreased, and as a result, the function of miR-151-3p is inhibited. As described in Examples below, when miR-151-3p is overexpressed, angiogenesis abnormalities are caused. Here, overexpression of miR-151-3p can be a state in which the secretion level or concentration of miR-151-3p found in malignant cancer cells is higher than that of normal cells or benign cancer cells. MiR-151-3p overexpression can also be caused by transfecting cells with miR-151-3p or miR-151-3p expression vectors. anti-miR-151-3p can suppress angiogenesis abnormalities due to such overexpression of miR-151-3p.
Here, the angiogenesis abnormality due to the overexpression of miR-151-3p refers to the promotion of angiogenesis due to the overexpression of miR-151-3p. The promotion of blood vessel formation can be confirmed using, for example, a known method such as the method described in the below-described examples, using as an index the increase in the length of the blood vessel and / or the number of branch points.

As used herein, “decoy RNA for miR-151-3p” has a partially complementary sequence to miR-151-3p, and is cleaved and degraded even after base pairing with miR-151-3p. RNA with a secondary structure that is difficult to receive. Decoy RNA is the same as anti-miR in that its function is inhibited by binding to target microRNA, but its structure is a hairpin RNA strand, and multiple target microRNAs are included in one molecule of decoy RNA. It differs from anti-miR in that it has an RNA binding sequence. 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 this, it is possible to avoid the decoy RNA cleavage by the RISC complex containing the target microRNA (Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells. Haraguchi T, Ozaki Y, Iba H. Nucleic Acids Res. 2009 Apr; 37 (6) e43).

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.

As another specific example of the present invention, “(a) a nucleotide sequence having 70% or more identity with the complementary strand sequence of miR-151-3p and inhibiting the function of miR-151-3p” Vectors designed to express a “nucleic acid comprising a nucleotide to do” can be mentioned. As used herein, the “expression vector” includes, for example, genetic information that can be replicated in a host cell and can be propagated autonomously, and can be isolated and purified from the host cell. Examples thereof include those in which the nucleic acid of (a) is introduced into an expression vector having a promoter that is easy and can function in a host cell and has a detectable marker. In the case of using a polIII promoter such as U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter as a promoter, the decoy RNA for anti-miR-151-3p and miR-151-3p The linker sequence and the gap sequence to avoid cleavage should be designed so that 4 or more U are not consecutive.

A commercially available miR-151-3p inhibitor can also be used. Anti-miR-151-3p is commercially available as Anti-miR ^™ miRNA inhibitor (Applied Biosystems), and commercially available decoy RNA against miR-151-3p is miArrest ^™ miRNA inhibitors (Catalog No. HmiR-AN0211- SN-5, -10, -20 (synthetic oligo), HmiR-AN0211-AM01, 02, 03, 04 (expression vector) (both manufactured by Genecopoeia)).

(D) The miR-886-3p inhibitor miR-886-3p is a molecule already known and typically means what is called mature miRNA. Here, miR-886-3p includes microRNA (miR-886-3p isomer) having the same sequence as miR-886-3p and present at different positions on the genome. Specifically, for example, it means a nucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8 (registered as Accession No. MIMAT0004906 in miRBase). The mature miR-886-3p means a single-stranded or double-stranded RNA consisting of the nucleotide sequence represented by SEQ ID NO: 8.

The miR-886-3p inhibitor may be any substance that inhibits the function of miR-886-3p, and its action mechanism is not limited.
“Functions of miR-886-3p”
(1) Activity to suppress the expression of a target gene having a nucleotide sequence complementary to miR-886-3p in mRNA (hereinafter, miR-886-3p target gene expression suppression activity), or (2) Cell angiogenesis Promoting activity,
Means.
“Inhibiting the function of miR-886-3p” means having the activity of suppressing the function of miR-886-3p, and whether to inhibit the function of miR-886-3p It can confirm by well-known methods, such as the method as described in an example.

Specific examples of miR-886-3p inhibitors include the following nucleic acids.
(A) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the complementary strand sequence of miR-886-3p, and comprising a nucleotide that inhibits the function of miR-886-3p, or (b) above (a) A nucleic acid that is an expression vector that expresses.
In the present invention, the complementary strand of miR-886-3p specifically refers to a nucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10.

“Nucleotide inhibiting the function of miR-886-3p” means that miR-886-3p is hybridized under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.) and miR- It means something that suppresses the function of 886-3p. More specifically, when miR-886-3p is hybridized under biological conditions (for example, 0.1 M phosphate buffer (pH 7.0) at 25 ° C.) and taken up into tumor cells, miR in the cells -886-3p means a nucleotide having the activity of suppressing the function of 3p.

The nucleotide sequence of “nucleotide that inhibits the function of miR-886-3p” used in the present invention is 70% or more, preferably 70% or more, preferably the nucleotide sequence represented by SEQ ID NO: 10, which is the complementary strand of miR-886-3p. 80% or more, more preferably 90% or more, and still more preferably 95% or more.

As the nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, one or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 10 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: 10 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: 10, and (3) a nucleotide represented by SEQ ID NO: 10. A 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: 10 A nucleotide sequence in which 1 to 6 (preferably 1 to 3, more preferably 1 or 2) nucleotides in the sequence are substituted with other nucleotides, or (5) the mutations (1) to (4) above Are combined nucleotide sequences (in this case, the sum of the 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: 10 is preferably 15 or more consecutive nucleotides (preferably 17 or more nucleotides or more, preferably included in the nucleotide sequence represented by SEQ ID NO: 10). A partial sequence of preferably 19 bases or more, most preferably 21 bases) or a sequence containing it.

Specific examples of the nucleic acid of the present invention include, for example, decoy RNA for anti-miR-886-3p and miR-886-3p.
As used herein, “anti-miR-886-3p” is a form of miR-886-3p microRNA activity-inhibiting molecule, and refers to a nucleic acid that is complementary to the miR-886-3p sequence.
Similar to the anti-miR-151-3p described above, the anti-miR-886-3p of the present invention may be modified so as to be resistant to various degrading enzymes.
As used herein, “decoy RNA for miR-886-3p” has a partially complementary sequence to miR-886-3p, and is cleaved and degraded even after base pairing with miR-886-3p. RNA with a secondary structure that is difficult to receive. Similar to the decoy RNA for miR-151-3p above, a sequence that is not completely complementary to the target microRNA (eg, 4 bases were inserted between the 10th and 11th bases from the 3 ′ end of the fully complementary sequence) Sequence) and the like, it is possible to avoid cleaving the decoy RNA by the RISC complex containing the target microRNA.

As another specific example of the present invention, “(a) a nucleotide sequence having 70% or more identity with the complementary strand sequence of miR-886-3p and inhibiting the function of miR-886-3p” Vectors designed to express a “nucleic acid comprising a nucleotide to do” can be mentioned. As used herein, the “expression vector” includes, for example, genetic information that can be replicated in a host cell and can be propagated autonomously, and can be isolated and purified from the host cell. Examples thereof include those in which the nucleic acid of (a) is introduced into an expression vector having a promoter that is easy and can function in a host cell and has a detectable marker. In addition, when using a polIII promoter such as U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter as a promoter, the above-mentioned decoy RNA for anti-miR-886-3p and miR-886-3p The linker sequence and the gap sequence to avoid cleavage should be designed so that 4 or more U are not consecutive.

A commercially available miR-886-3p inhibitor can also be used. Anti-miR-886-3p is commercially available as Anti-miR ^TM miRNA inhibitor (Applied Biosystems), and miRrest ^TM miRNA inhibitors (catalog No. HmiR-AN0813- SN-5, -10, -20 (synthetic oligo), HmiR-AN0813-AM01, 02, 03, 04 (expression vector) (both manufactured by Genecopoeia)).

(E) SMPD3 inhibitor The SMPD3 inhibitor may be any substance that suppresses the expression of SMPD3 or a substance that suppresses the function of SMPD3, and its action mechanism is not limited. SMPD3 is known to regulate the secretion of microRNA in membrane vesicles called exosomes (J Biol Chem. 2010 Jun 4; 285 (23): 17442-52).
A substance that suppresses the expression or function of SMPD3 is useful for tumor therapy because it suppresses tumor metastasis by suppressing tumor angiogenesis.

SMPD3 is a known molecule and means a neutral sphingomyelinase 2. SMPD3 is a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 12. In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation.
Specifically, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 12 (RefSeq No. NP_061137), or an ortholog thereof (for example, mouse (RefSeq No. NP_067466), rhesus monkey ( RefSeq No. XP_001091683), dogs (RefSeq No. XP_546863), rats (RefSeq No. NP_446057), cows (RefSeq No. NP_001179292), etc.), and their splice variants and allelic variants.
SMPD3 is a human or other warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit, dog, pig, sheep, cow, monkey, etc.) cell (eg, MCF7 cell, vascular cell, brain cell) or those May be isolated and purified from any tissue (eg, mammary gland tissue, small pulmonary artery, brain) and the like by a known protein separation and purification technique.

“Amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 12”
(a) an amino acid sequence having about 80% or more homology with the amino acid sequence represented by SEQ ID NO: 12;
(b) in the amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence in which 1 to 50 amino acids are substituted and / or deleted and / or inserted and / or added;
(c) the amino acid sequence of an ortholog in another mammal of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 12; or
(d) The amino acid sequence of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 12 or the orthologue splice variant, allelic variant or polymorphism of (c) above.

As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

The homology of the amino acid sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms include NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 1997 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [The algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0)], Pearson et al., C Proc. Natl. Acad. Sci. USA, 85: 2444-2448 1988 (1988) [the algorithm is a GCG software package. Embedded in the FASTA program in the cage] and the like, and these can be preferably used as well.

In the above (a), more preferably, the “amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 12” is about 80% or more of the amino acid sequence represented by SEQ ID NO: 12; The amino acid sequence preferably has about 90% or more, more preferably about 95% or more, still more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more.

“A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 12” includes an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 12 It is a protein having substantially the same activity as the protein consisting of the amino acid sequence represented by No. 12
Here, “activity” refers to the activity of hydrolyzing the phosphodiester bond of sphingomyelin. Further, “substantially the same quality” means that the properties are qualitatively the same, for example, physiologically or pharmacologically.
The sphingomyelin hydrolyzing activity can be measured by measuring and comparing the amount of phosphorylcholine produced by hydrolyzing sphingomyelin according to a method known per se. For example, it can be measured using a commercially available Sphingomyelinase Assay kit (Cayman).

In addition, as SMPD3 in the present invention, as shown in (b) above, for example, (i) 1 to 50, preferably 1 to 30, more preferably 1 to 1 in the amino acid sequence represented by SEQ ID NO: 12 Amino acid sequence in which 10 amino acids, more preferably 1 to several (5, 4, 3 or 2) amino acids have been deleted, (ii) 1 to 50 amino acid sequences represented by SEQ ID NO: 12, preferably 1 To 30 amino acids, more preferably 1 to 10, more preferably 1 to several (5, 4, 3 or 2) amino acids, (iii) the amino acid sequence represented by SEQ ID NO: 12 An amino acid sequence in which 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably 1 to several (5, 4, 3 or 2) amino acids are inserted, (iv) SEQ ID NO: : 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably in the amino acid sequence represented by 12 Also included are so-called muteins such as proteins containing amino acid sequences in which one to several (5, 4, 3, or 2) amino acids are substituted with other amino acids, or (v) amino acid sequences combining them.
When the amino acid sequence is inserted, added, deleted or substituted as described above, the position of the insertion, addition, deletion or substitution is not particularly limited as long as the sphingomyelin hydrolysis activity is maintained.

In the present invention, the “substance that suppresses the expression of SMPD3” acts at any stage such as the transcription level of SMPD3 gene encoding SMPD3, the level of post-transcriptional regulation, the level of translation into SMPD3, the level of post-translational modification, etc. It may be. Therefore, examples of substances that suppress SMPD3 expression include substances that inhibit transcription of the SMPD3 gene (eg, antigenes), substances that inhibit the processing of early transcripts into mRNA, and those that inhibit mRNA transport to the cytoplasm. Substance that inhibits translation from mRNA to SMPD3 (eg, antisense nucleic acid, miRNA) or degrades mRNA (eg, siRNA, ribozyme, miRNA), substance that inhibits post-translational modification of the initial translation product, etc. Is included. Any substance that acts at any stage can be preferably used, but a substance that binds complementarily to mRNA and inhibits translation into SMPD3 or decomposes mRNA is preferable.

As a substance that specifically inhibits translation of SMPD3 gene from mRNA to SMPD3 (or degrades mRNA), preferably a base sequence complementary to or substantially complementary to the base sequence of these mRNAs or a part thereof The nucleic acid containing is mentioned.
The base sequence substantially complementary to the base sequence of the mRNA of the SMPD3 gene can bind to the target sequence of the mRNA and inhibit its translation under physiological conditions in mammals (or cleave the target sequence). A base sequence having a degree of complementarity, specifically, for example, a region that overlaps with a base sequence that is completely complementary to the base sequence of the mRNA (that is, a base sequence of the complementary strand of the mRNA). The nucleotide sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 97% or more.
The “base sequence homology” in the present invention uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gaps; filtering = ON; It can be calculated by match score = 1; mismatch score = -3).

More specifically, the base sequence complementary or substantially complementary to the base sequence of the mRNA of the SMPD3 gene is (a) complementary or substantially complementary to the base sequence represented by SEQ ID NO: 11. Or (b) a base sequence that hybridizes with a complementary strand of the base sequence represented by SEQ ID NO: 11 under stringent conditions, comprising the amino acid sequence represented by SEQ ID NO: 12. Examples of the sequence that encodes a protein having substantially the same quality of activity as a protein and complementary or substantially complementary base sequences. Here, “substantially the same quality of activity” has the same meaning as described above.

The stringent conditions are, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley and Sons, 6.3.1-6.3.6, 1999, for example, 6 × SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washes at 0.2 × SSC / 0.1% SDS / 50 to 65 ° C., those skilled in the art will know the conditions for hybridization that will give the same stringency. It can be selected appropriately.

As a preferred example of mRNA of the SMPD3 gene, a human SMPD3 gene (RefSeq No. NM_018667) containing the nucleotide sequence represented by SEQ ID NO: 11 or an ortholog thereof (for example, mouse (RefSeq No. NM_021491) in other mammals. ), Rhesus monkeys (RefSeq No. XM_001091683), dogs (RefSeq No. XM_546863), rats (RefSeq No. NM_053605), cattle (RefSeq No. NM_001192363)), and their splice variants, allelic variants, polymorphisms, etc. Of mRNA.

“Part of the base sequence complementary to or substantially complementary to the base sequence of the mRNA of the SMPD3 gene” means that it can specifically bind to the mRNA of the SMPD3 gene and translates the protein from the mRNA. The length and the position are not particularly limited as long as they can inhibit (or degrade the mRNA), but at least 10 bases that are complementary or substantially complementary to the target sequence from the viewpoint of sequence specificity. As mentioned above, it contains about 15 bases or more, more preferably about 20 bases or more.

Specifically, the nucleic acid containing any one of the following (a) to (c) is preferably exemplified as a nucleic acid complementary to or substantially complementary to the nucleotide sequence of the mRNA of the SMPD3 gene or a part thereof: Is done.
(a) Antisense nucleic acid against mRNA of SMPD3 gene
(b) Ribozyme nucleic acid for mRNA of SMPD3 gene
(c) a nucleic acid having RNAi activity against mRNA of the SMPD3 gene or a precursor thereof

(a) Antisense nucleic acid against SMPD3 gene mRNA The “antisense nucleic acid against SMPD3 gene mRNA” in the present invention includes a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. It is a nucleic acid and has a function of suppressing protein synthesis by forming a specific and stable duplex with a target mRNA.
Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base such as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-rigid Having a side chain group such as sugar (eg, monosaccharide), having an intercurrent compound (eg, acridine, psoralen), chelate compound (eg, metal, having radioactivity) It may be one containing a metal, boron, oxidizing metal, etc., one containing an alkylating agent, or one having a modified bond (for example, α-anomeric nucleic acid etc.). Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted.

As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the SMPD3 gene. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the SMPD3 gene using a homology search program such as BLAST or FASTA.

The length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, the translation into SMPD3 is inhibited. MRNA encoding SMPD3 The short sequence may be about 10 bases, and the long sequence may be the entire mRNA or initial transcription product sequence. In view of easiness of synthesis, antigenicity, intracellular migration, etc., an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto. Specifically, 5 'end hairpin loop of SMPD3 gene, 5' end 6-base pair repeat, 5 'end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region , 3 ′ end palindromic region or 3 ′ end hairpin loop, etc. may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.

Furthermore, the antisense nucleic acid of the present invention not only hybridizes with the mRNA of the SMPD3 gene and the initial transcription product to inhibit translation into protein, but also binds to these genes that are double-stranded DNA to form triple strands ( A triplex) that can inhibit transcription to RNA (antigene).

The nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the 2′-position hydroxyl group of each sugar (ribose) is changed to OR (R═CH ₃ (2′-O-Me), F (2′-F), CH ₂ CH ₂ OCH ₃ (2′- O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.

The conformation of the sugar part of nucleic acids is dominated by C2'-endo (S-type) and C3'-endo (N-type). In single-stranded nucleic acids, they exist as an equilibrium between them, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, it is a nucleic acid derivative in which the 2 'oxygen and 4' carbons are cross-linked via a methylene residue to fix the sugar conformation to the N type. LNA (Imanishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and 2 'oxygen and 4' carbon with ethylene residues ENA (Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003), which is a nucleic acid derivative in which the conformation of the sugar moiety is fixed to the N-type by crosslinking via the Can be used.

The antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of SMPD3 gene, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman) Etc.) can be prepared by synthesizing a complementary sequence thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.

(b) Ribozyme nucleic acid for mRNA of SMPD3 gene As another preferred example of a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of mRNA of SMPD3 gene or a part thereof, the mRNA is used as a coding region. Examples include ribozyme nucleic acids that can be cleaved specifically inside. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When the mRNA of the SMPD3 gene has a double-stranded structure itself, the target sequence is made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

(c) Nucleic acid having RNAi activity against mRNA of SMPD3 gene or a precursor thereof In this specification, double-stranded RNA consisting of oligo RNA complementary to mRNA of SMPD3 gene and its complementary strand, so-called siRNA is also included. Further, it is defined as being included in a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of SMPD3 gene or a part thereof. When a short double-stranded RNA is introduced into a cell, the mRNA complementary to that RNA is degraded. So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Since this phenomenon has been confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme.

SiRNA can be designed according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)) based on the cDNA sequence information of the target gene. Examples of siRNA target sequences include, but are not limited to, AA + (N) 19, AA + (N) 21 or NA + (N) 21 (N is an arbitrary base). The position of the target sequence is not particularly limited. For the selected target sequence candidate group, whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) And the like, and the specificity of the selected target sequence is confirmed. For example, when AA + (N) 19, AA + (N) 21 or NA + (N) 21 (N is an arbitrary base) is used as the target sequence, the target sequence whose specificity has been confirmed is AA (or NA) or later. Two strands consisting of a sense strand having a TT or UU 3 'end overhang at 19-21 bases and an antisense strand having a sequence complementary to the 19-21 base and a TT or UU 3' end overhang Strand RNA may be designed as siRNA. In addition, for short hairpin RNA (shRNA) which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are combined with each other. It can be designed by linking via a linker sequence.

The siRNA and / or shRNA sequences can be searched using search software provided free of charge on various websites. Examples of such sites include siRNAsiTarget Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and pSilencerTM Expression Vector insert design tools (http: // www.ambion.com/techlib/misc/psilencer_converter.html), GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex, but not limited to these.

The ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like.

The siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, it can be prepared by synthesizing a single hairpin RNA (shRNA) serving as a siRNA precursor and cleaving it with a dicer.

In the present specification, a nucleic acid designed to generate an siRNA against the mRNA of the SMPD3 gene in vivo is also a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the mRNA of the SMPD3 gene. Defined as encompassed by a nucleic acid containing a moiety. Examples of such nucleic acids include expression vectors constructed so as to express the above-mentioned shRNA and siRNA. shRNA is an oligo containing a base sequence in which the sense strand and the antisense strand of the target sequence on mRNA are linked by inserting a spacer sequence (for example, about 5 to 25 bases) long enough to form an appropriate loop structure. It can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer. Vectors expressing shRNA include tandem type and stem loop (hairpin) type. In the former, siRNA sense and antisense strand expression cassettes are linked in tandem, and each strand is expressed and annealed in the cell to form a double-stranded siRNA (dsRNA). It is. On the other hand, the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA. As a promoter, a pol II promoter (for example, a CMV immediate early promoter) can be used, but in order to perform transcription of a short RNA accurately, a pol III promoter is generally used. Examples of the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, and the like. Further, a sequence in which 4 or more Ts are continuous is used as a transcription termination signal.
The siRNA or shRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector. Examples of such vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids, and the like.

Nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the SMPD3 gene or a part thereof is provided in a special form such as a liposome or a microsphere, applied to gene therapy, It can be given in an added form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of nucleic acids and can be attached via bases, sugars, intramolecular nucleoside linkages. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

The SMPD3 expression-suppressing activity of these nucleic acids can be examined using a transformant introduced with an SMPD3 gene, an SMPD3 gene expression system in vivo or in vitro, or an SMPD3 translation system in vivo or in vitro.

The substance that suppresses the expression of SMPD3 in the present invention is not limited to a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the SMPD3 gene as described above or a part thereof, and SMPD3 production Other substances such as low molecular weight compounds may be used as long as they are directly or indirectly inhibited. Such a substance can be obtained, for example, by the screening method of the present invention described later.

In the present invention, the “substance that suppresses the function of SMPD3” means that once functionally produced SMPD3 suppresses angiogenesis, suppresses microRNA secretion, or suppresses tumor metastasis. Any thing is acceptable. Whether or not to suppress the function of SMPD3 can be confirmed by a known method such as the method described in Examples. Examples of a substance that suppresses the function of SMPD3 include a substance that binds to SMPD3 and suppresses tumor angiogenesis, GW4869 (3,3 ′-(1,4-Phenylene) bis [N- [4- (4, Examples include substances that inhibit sphingomyelin hydrolysis such as 5-dihydro-1H-imidazol-2-yl) phenyl] -2-propenamide]).

Specifically, examples of the substance that suppresses the function of SMPD3 include an antibody against SMPD3. The antibody may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known antibody or antiserum production methods. The isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen. In addition to a complete antibody molecule, for example, Fab, Fab ′, F (ab ') 2 fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having protein stabilizing action such as polyethylene glycol (PEG) They may be modified derivatives thereof.
In a preferred embodiment, since the antibody against SMPD3 is used as a pharmaceutical for human administration, the antibody (preferably a monoclonal antibody) is an antibody with reduced risk of showing antigenicity when administered to humans. Specific examples include fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, and particularly preferably fully human antibodies. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods. In addition, fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to provide a large amount of antibodies stably and at low cost, human antibody-producing mice and phage display methods are used. It is desirable to manufacture using.

Since SMPD3 plays an important role in tumor metastasis, especially in the process of cancer cell overflow from the primary lesion to the bloodstream, by controlling tumor angiogenesis, substances that inhibit SMPD3 function as cell membranes. It is desirable that the material has excellent permeability. Therefore, a more preferable substance that suppresses the function of SMPD3 is a low molecular compound suitable for Lipinski's Rule. Such a compound can be obtained, for example, using the screening method of the present invention described later.

(1-1) Pharmaceutical containing the nucleic acid, siRNA and precursor thereof of the present invention The agent of the present invention comprises an effective amount of the nucleic acid of any one of (A) to (D) above or the expression of SMPD3 of (E) above In addition to the nucleic acid that inhibits, any carrier, such as a pharmaceutically acceptable carrier, can be included and 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 agent of the present invention into tumor cells, the agent of the present invention can further contain a reagent for nucleic acid introduction. The nucleic acid introduction reagent includes atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectum), 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 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.

(1-2) Drugs containing SMPD3 antibodies, low molecular compounds that suppress SMPD3 expression or function, etc. Antibodies to SMPD3 and low molecular compounds that suppress SMPD3 expression or function inhibit SMPD3 production or activity can do. Accordingly, these substances suppress the expression or function of SMPD3 in vivo and suppress tumor angiogenesis or metastasis, and thus can be used as a prophylactic and / or therapeutic agent for tumors.
The medicine containing the above antibody or low molecular weight compound can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey). Etc.) orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).

The above-described antibodies and low-molecular compounds may be administered per se, or may be administered as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the above antibody or low molecular compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.

As a composition for parenteral administration, for example, injection, suppository, intranasal administration, etc. are used, and the injection is intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection. You may include dosage forms, such as an agent. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared by, for example, dissolving, suspending or emulsifying the antibody or low molecular compound of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository used for rectal administration may be prepared by mixing the above-described antibody or a salt thereof with an ordinary suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The antibody or low molecular weight compound is preferably contained in an amount of usually 0.1 to 500 mg, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms per dosage unit form.

The dose of the above-mentioned medicament containing the above-mentioned antibody or low-molecular compound or a salt thereof varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for treatment / prevention of tumors Is usually about 0.0001 to 20 mg / kg body weight for a single dose of antibody or low molecular weight compound, about 1 to 5 times a day for low molecular weight compound, orally or parenterally, 1 to several days for antibody Conveniently administered once a month by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

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, the tumor to which the agent of the present invention can be applied is preferably cancer, and more preferably breast cancer.
Since the agent of the present invention has an activity of suppressing angiogenesis, tumors can be treated by administering the agent of the present invention to cancer patients, particularly breast cancer patients. Moreover, since it has an activity of suppressing angiogenesis, it can suppress tumor metastasis.
Therefore, the agent of the present invention is extremely useful as a therapeutic agent for tumors.

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 concomitant drug of the present invention is not particularly limited, but preferably has an activity of suppressing the growth of the tumor itself. 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, etoposiderstat, gefinitib, 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.
As an administration subject, a breast cancer patient can be mentioned preferably. In addition, the relative levels of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p or miR-886-3p Patients with high cancer are desirable.

The dosage of the active ingredient of the agent 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). is there.

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 dosage form of the agent of the present invention and the antitumor agent is not particularly limited as long as the agent of the present invention and the antitumor agent are combined at the time of administration. Examples of such dosage forms include:
(1) Administration of a single preparation obtained by simultaneously formulating the agent of the present invention and an antitumor agent,
(2) Simultaneous administration by the same administration route of two types of preparations obtained by separately formulating the agent of the present invention and the antitumor agent,
(3) Administration of the two preparations obtained by separately formulating the agent of the present invention and the antitumor agent with a time difference in the same administration route,
(4) Simultaneous administration of two kinds of preparations obtained by separately formulating the agent of the present invention and the antitumor agent through different administration routes,
(5) Administration of the two preparations obtained by separately formulating the agent of the present invention and the antitumor agent at different time intervals in different administration routes (for example, in the order of the agent of the present invention → the antitumor agent) Administration, or administration in the reverse order).

An agent comprising the agent 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 agent 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-mentioned agent of the present invention and the antitumor agent are separately formulated and administered together, the agent of the present invention and the antitumor agent may be administered at the same time, but the antitumor agent is administered first. Thereafter, the agent of the present invention may be administered, or the agent of the present invention may be administered first, followed by administration of the antitumor agent. 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 administering an antitumor agent first, after administration of a preparation containing the antitumor agent, Examples include a method of administering the agent of the present invention within 3 days, preferably within 10 minutes to 1 day, more preferably within 15 minutes to 1 hour. When the agent of the present invention is administered first, the antitumor agent is administered within 1 minute to 1 day, preferably within 10 minutes to 6 hours, more preferably within 15 minutes to 1 hour after the administration of the agent of the present invention. The method of administration 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 concomitant drug of the present invention is preferably applied to breast cancer.

3. The present invention relates to miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886 in test samples. -3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280 , Based on measuring the secretion level or concentration of miR-210, miR-140-3p, miR-593 or miR-483-3p and a positive correlation between the secretion level or the concentration and malignant cancer Providing a method for determining malignant cancer, comprising determining whether the cancer is malignant. The method of the present invention is particularly useful for the determination of malignant breast cancer.
As used herein, “malignant cancer” refers to a cancer that has higher tumor forming ability, higher metastatic ability, and higher patient mortality than benign cancer.

A test sample is a cell, blood 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 is preferably a mammary gland, and more preferably a mammary gland cell.
In the determination method of the present invention, miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886 in the test sample -3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280 , MiR-210, miR-140-3p, miR-593 or miR-483-3p secretion levels or concentrations are measured.

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

For example, miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR-140-3p, The secretion level or concentration of miR-593 or miR-483-3p can be measured by a method known per se using a nucleic acid probe capable of specifically detecting 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-29b-1 * is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most contained in the nucleotide sequence represented by SEQ ID NO: 1. Preferably, the polynucleotide includes a full-length continuous nucleotide sequence or a complementary sequence thereof.

The nucleic acid probe capable of specifically detecting miR-221 * 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: 2. Mention may be made of polynucleotides comprising the full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-138 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 3. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-584 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 4. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-30a is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 5. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-146a is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 6. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-151-3p 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: 7. May include a polynucleotide comprising its full length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-886-3p 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: 8. May include a polynucleotide comprising its full length contiguous nucleotide sequence or its complementary sequence.

As a nucleic acid probe capable of specifically detecting miR-100, 15 nucleotides or more, preferably 18 nucleotides or more, more preferably about 20 nucleotides or more, most preferably, included in the nucleotide sequence represented by SEQ ID NO: 13 Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-221 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, and most preferably the nucleotide sequence represented by SEQ ID NO: 14. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-126 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 15. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-130a is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 16. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-222 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence contained in the nucleotide sequence represented by SEQ ID NO: 17. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-125b is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 18. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-29a is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 19. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

As a nucleic acid probe capable of specifically detecting miR-720, it is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 20. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-224 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence contained in the nucleotide sequence represented by SEQ ID NO: 21. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-29b is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 22. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-1274b is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 23. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-1280 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 24. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-210 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 25. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-140-3p 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: 26. May include a polynucleotide comprising its full length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-593 is 15 bases or more, preferably 18 bases or more, more preferably about 20 bases or more, most preferably the nucleotide sequence represented by SEQ ID NO: 27. Mention may be made of polynucleotides comprising a full-length contiguous nucleotide sequence or its complementary sequence.

The nucleic acid probe capable of specifically detecting miR-483-3p 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: 28. May include a polynucleotide comprising its 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, ³² P, ³³ P, ³⁵ S, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), etc. It may be labeled with. 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 a DNA / RNA automatic synthesizer based on the information of the nucleotide sequence represented by SEQ ID NOs: 1 to 8 or 13 to 28, for example.

Then measured miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR- Based on the secretion level or concentration of 140-3p, miR-593 or miR-483-3p, it is determined whether the test sample is malignant cancer.
As shown in the examples below, malignant cancer cells are miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a compared to normal cells and benign cancers. , MiR-151-3p, miR-886-3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR -29b, miR-1274b, miR-1280, miR-210, miR-140-3p, miR-593 and miR-483-3p have high secretion levels or concentrations.
Therefore, the above judgment is miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR- Based on a positive correlation between the secretion level or concentration of 140-3p, miR-593 or miR-483-3p and malignant cancer.

For example, cells or tissues collected from non-cancer or benign cancer patients (negative control) and malignant cancer patients (positive control), and miR-29b-1 *, miR-221 *, MiR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221, miR-126, miR-130a, miR-222, Secretion levels of miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR-140-3p, miR-593 or miR-483-3p Is compared to that of the positive and negative controls. Alternatively, miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR- in specific cells or tissues 100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, A correlation diagram between the secretion level of miR-140-3p, miR-593 or miR-483-3p and malignant cancer is prepared in advance, and the secretion level in the test sample collected from the target patient is compared with the correlation diagram May be. The comparison of the secretion level is preferably performed based on the presence or absence of a significant difference.
From the comparison results of the secretion level, the measurement target miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886- 3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, If the secretion level of miR-210, miR-140-3p, miR-593 or miR-483-3p is relatively high, determine that the test sample is relatively likely to have malignant cancer Can do. Conversely, the measurement target miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR- When the secretion level of 140-3p, miR-593 or miR-483-3p is relatively low, it can be determined that the possibility that the test sample is malignant cancer is relatively low.

The therapeutic target of the agent of the present invention includes miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p or miR in the test sample. Since cancer patients with a relatively high secretion level of -886-3p are desirable, the methods of the present invention are useful for patient screening.

The present invention also includes miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR- 100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, An agent for determining the presence or malignancy of cancer (hereinafter referred to as “the agent of the present invention”) comprising a nucleic acid probe capable of specifically detecting miR-140-3p, miR-593 or miR-483-3p (II) ").). The agent (II) of the present invention can be a kit for determining the presence or absence of malignant cancer in a test sample or the degree of malignancy. By using the agent (II) of the present invention, it is possible to easily determine whether the test sample has malignant cancer 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 phase carrier. 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 is miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR -100, miR-221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210 Depending on the measurement method of miR-140-3p, miR-593 or miR-483-3p, the composition may further contain other components necessary for carrying out the method. 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 the measurement, the agent (II) of the present invention can further contain a labeling reagent, a chromogenic substrate, and the like.

4). The present invention relates to a method of searching for a substance capable of suppressing tumor angiogenesis.The present invention relates to the secretion of a test substance miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a. Alternatively, the present invention provides a method for searching for a substance that suppresses angiogenesis, including evaluating whether to enhance expression, and a substance that can be obtained by the method.
In the search method of the present invention, a substance that upregulates the secretion or expression of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a suppresses angiogenesis Drug, ie, a substance capable of suppressing tumor metastasis or a tumor therapeutic agent.

The search method of the present invention includes the following steps:
(1) contacting a test substance with a cell capable of measuring secretion or expression of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a;
(2) measuring the amount of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a secreted or expressed in cells contacted with the test substance, Comparing the amount of secretion or expression with the amount of secretion or expression in control cells not contacted with the test substance; and (3) based on the comparison result of (2) above, miR-29b-1 *, miR-221 * Select a test substance that up-regulates the secretion or expression level of miR-138, miR-584, miR-30a, or miR-146a as a substance that can suppress tumor angiogenesis.

The present invention also provides a substance that suppresses angiogenesis, comprising evaluating whether a test substance suppresses the secretion, expression level or function of miR-151-3p, miR-886-3p or SMPD3. Provided are a method for searching and a substance obtainable by the method.
In the search method of the present invention, the substance that down-regulates the secretion or function of miR-151-3p, miR-886-3p or SMPD3 is an agent that suppresses angiogenesis, that is, a substance or tumor that can suppress tumor metastasis Selected as a therapeutic agent.

The search method of the present invention includes the following steps:
(1) contacting a test substance with a cell capable of measuring secretion or expression of miR-151-3p, miR-886-3p or SMPD3;
(2) Measure the secretion amount, expression level or function of miR-151-3p, miR-886-3p or SMPD3 in cells contacted with the test substance, and determine the secretion amount, expression level or function of the test substance Comparing with the secreted amount, expression level or function in the non-contacted control cells; and (3) secreted amount, expression of miR-151-3p, miR-886-3p or SMPD3 based on the comparison result of (2) above A test substance that down-regulates the amount or function is selected as a substance capable of suppressing tumor angiogenesis.

In addition, the present invention provides a dual VEGF signal and its feedback signal, the Akt3-DLL4 pathway, comprising evaluating whether a test substance suppresses the secretion, expression level or function of miR-151-3p. The present invention provides a method for searching for a substance that suppresses abnormal angiogenesis by inhibiting it as well as a substance that can be obtained by the method.
In the search method of the present invention, a substance that down-regulates the secretion or function of miR-151-3p is selected as an agent that suppresses angiogenesis abnormality, that is, a substance that can suppress tumor metastasis or a tumor therapeutic agent.
In this case, the search method of the present invention may further include a step of measuring the expression level of Akt3 and / or DLL4. The expression level of Akt3 and / or DLL4 can be measured by a method known per se using, for example, an antibody that specifically recognizes Akt3 and / or DLL4.

The search method of the present invention includes the following steps:
(1) contacting a test substance with a cell capable of measuring secretion or expression of miR-151-3p;
(2) measuring the secretion amount, expression amount or function of miR-151-3p in a cell contacted with a test substance, and the secretion amount, expression amount or function in a control cell not contacting the test substance; Comparing with the expression level or function; and (3) based on the comparison result of (2) above, a test substance that down-regulates the secretion amount, expression level or function of miR-151-3p, Select as a substance that can suppress abnormalities.

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.

In the search method of the present invention, microRNAs (miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151 whose secretion level or expression level is measured are measured. -3p or miR-886-3p) include mature, pri-miRNA and pre-miRNA, but preferably the sum of all these types of secretion levels or expression levels or mature secretion levels or expression The level, more preferably the mature secretion level or expression level, is measured.

`` Cells capable of measuring secretion or expression '' are miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, A cell capable of evaluating the secretion level or expression level of miR-886-3p or SMPD3. The cells include miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p or SMPD3 to be measured. Cells that can be secreted or expressed in nature are mentioned.

Naturally secreted or measured, i.e. miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p or SMPD3 Cells capable of expression are potentially miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p or SMPD3 The cell is not particularly limited as long as it is secreted or expressed, and as the cell, a primary cultured cell of a mammal (eg, human, mouse, etc.), a cell line derived from the primary cultured cell, or the like can be used. Cells capable of naturally secreting or expressing miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p or SMPD3 Examples thereof include tumor cells detailed in the above section (1. Agent of the present invention) as “Tumors to which the agent of the present invention can be applied”. Breast cancer cells are preferred, and malignant breast cancer cells are more preferred.

Secretion or expression of the test substance and miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p or SMPD3 Contact with measurable cells takes place in the culture medium. Culture medium can measure miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p or SMPD3 secretion For example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified Eagle medium (DMEM), or the like is selected. 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.

Measurement of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p or SMPD3 , (2. Method for determining cancer) can be performed according to a known method such as the method described in the section.
The function level of SMPD3 can be measured by evaluating the function of the known SMPD3 described in the section (1. Agent of the present invention).

The comparison of the secretion amount or the expression amount can be preferably performed based on the presence or absence of a significant difference. MiR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p in control cells not contacted with the test substance Alternatively, the amount of SMPD3 secreted or expressed is miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151 in cells contacted with the test substance. -3p, miR-886-3p or SMPD3 secretion amount or expression level measurement may be a secretion amount or expression level measured in advance, or simultaneously measured secretion amount or expression level, From the viewpoint of the accuracy and reproducibility of the experiment, the amount of secretion or expression measured simultaneously is preferable.

Then, miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a obtained as a result of the comparison up-regulated, or miR- Substances that down-regulate the secretion, expression, or function of 151-3p, miR-886-3p or SMPD3 are substances that can suppress angiogenesis, especially those that can inhibit tumor angiogenesis, or tumor metastasis It is selected as a substance that can be suppressed.

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

In the sequence listing of the present application, 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 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.

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

Reference Example 1 Preparation of firefly luciferase stable expression 4T1 cells (4T1-luc cells) Using 1 μg of pNeoLuc containing firefly luciferase gene, using LipofectamineLTX kit (Invitrogen) according to the procedure attached to the reagent kit. ^The cells were introduced into 4T1 cells (ATCC CRL-2539), a mouse breast cancer cell line seeded at a concentration of ⁶ cells / well (6-well plate). On the next day, the medium was changed to a 10% FBS / RPMI medium (Invitrogen) containing 0.5 mg / mL Geneticin (Invitrogen), and the cells were stably cultured for 2 weeks to select stably expressing cells. Firefly luciferase activities of a plurality of stably expressing cells were measured using Bright Glo reaction solution (Promega) and Envision 2101 MultiLabel Reader (Wallac), and the most active cells were selected. As a result, it was possible to establish cells exhibiting a luminescence amount of about 3,000 RLU per 10,000 cells. Thereafter, the cell line was used as firefly luciferase stably expressing 4T1 cells (4T1-luc cells).

Reference Example 2 Preparation of SMPD3 Knockdown 4T1-luc Cells Using 1 μg of plasmid pBAsi-mU6 Pur (Takara Bio TA0715-1-C) containing the siRNA of mouse SMPD3 gene (GTACATCCTGTATGATGTT) using LipofectamineLTX kit (Invitrogen) According to the procedure attached to the reagent kit, the cells were introduced into 4T1-luc cells established by the method described in Example 1 seeded at a concentration of 1 × 10 ⁶ cells / well (6-well plate). On the next day, the medium was replaced with 10% FBS / RPMI medium (Invitrogen) containing 2 μg / mL Puromycin (Invitrogen), and the cells were stably cultured for 2 weeks to select stably expressing cells. The expression level of mouse SMPD3 gene in a plurality of stably expressing cells was quantified using mouse SMPD3 TaqMan Reagent (Applied Biosystems). First, total RNA was prepared from stably expressing cells using RNeasy mini kit (Qiagen). Next, cDNA synthesis was performed based on the total RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). That is, mix 0.5 μg total RNA, 1.0 μL 10 × RT buffer, 0.4 μL 25 × dNTP, 1.0 μL 10 × RT Primer, 0.5 μL Transcriptase, 0.5 μL RNase Inhibitor, make up to 10 μL with distilled water, CDNA synthesis was carried out by carrying out a heat retention step of 25 ° C. for 10 minutes, 37 ° C. for 2 hours, and 85 ° C. for 5 seconds. Next, TaqMan RT-PCR reaction was performed using the cDNA as a template. That is, 2.25 μL of the cDNA solution, 5 μL of Platinum qPCR SuperMix-UDG with ROX (Invitrogen), 0.5 μL of mSMPD3 TaqMan reagent (Applied Biosystems) or mouse β-actin TaqMan reagent (Applied Biosystems) are mixed. The volume was made up to 10 μL with distilled water. The reaction mixture was incubated at 95 ° C for 2 minutes using ABI7300 (Applied Biosystems), and then the incubation cycle of 95 ° C for 15 seconds and 60 ° C for 30 seconds was repeated 45 times to quantify mouse SMPD3 and mouse β-actin. Went. The mouse SMPD3 expression level / mouse β-actin expression level of each stable expression cell line was calculated, and the stable expression cell line (4T1-luc mSMPD3 KD cell) having the highest calculated value was used in the following experiment.

Example 1 Effect of SMPD3 Knockdown on Metastasis Assay in a Subcutaneous Transplant Model 1 × 10 ⁶ 4T1-luc mSMPD3 suspended in PBS subcutaneously in both inguinal regions of 5 7-week-old female Balb / c athymic nude mice KD cells were transplanted. As a control group, Balb / cathic nude mice transplanted with 1 × 10 ⁶ 4T1-luc cells were used. 14 days after transplantation, luciferase activity in lungs extracted from mice was measured using in vivo imaging, and it was found that lung metastasis in mice transplanted with 4T1-luc mSMPD3 KD cells was significantly reduced compared to the control group. I understand (Figure 1).

Example 2 Effect of SMPD3 Knockdown on Metastasis Assay in Tail Vein Administration Model 1 × 10 ⁶ 4T1-luc mSMPD3 KD suspended in PBS from the tail vein of five 7-week-old female Balb / c athymic nude mice Cells were transplanted. As a control group, Balb / cathic nude mice transplanted with 1 × 10 ⁶ 4T1-luc cells were used. Twenty-six days after transplantation, luciferase activity in lungs isolated from mice was measured using in vivo imaging, and it was found that lung metastasis in mice transplanted with 4T1-luc mSMPD3 KD cells was similar to that in the control group (FIG. 2). ). This suggests that SMPD3 knockdown 4T1-luc cells have suppressed the process of overflow from the primary lesion to the bloodstream.

Example 3 Effect of SMPD3 Knockdown on Tumor Angiogenesis in a Subcutaneous Transplant Model 4T1-luc mSMPD3 Tumor masses in the inguinal region of Balb / cathic nude mice 20 days after transplantation of KD cells were extracted and fixed in formalin. After that, 5 μm paraffin-embedded sections were immunostained with the antibody against CD31, a vascular endothelium-specific protein (Rabbit polyclonal to CD31 ab28364 (Abcam)), according to the following procedure, and the ability to form tumor blood vessels was confirmed. evaluated. As a control group, a tumor mass at the groin part of Balb / cathic nude mice transplanted with 4T1-luc cells was used. First, the section was washed with xylene three times for 5 minutes, and then washed for 5 minutes in the order of 99.5% ethanol, 80% ethanol, and 70% ethanol. Further, after washing 3 times with distilled water for 5 minutes, it was immersed in running water for 10 minutes. Subsequently, after washing 3 times with distilled water for 5 minutes, autoclaving was performed at 121 ° C. for 5 minutes in 0.5 M Tris buffer (pH = 10). Then, after cooling by standing at room temperature for 30 minutes, washed with distilled water for 5 minutes, 3 times, followed by TBS-T solution (0.05M Tris, 0.3M NaCl, 0.1% Tween20 aqueous solution) for 5 minutes. Washed twice. After surrounding the tissue with a super pap pen, it was immersed in Image-iT Ex Signal Enhancer (Molecular probe) at room temperature for 30 minutes. After washing with TBS-T solution, it was immersed in Dako Protein Block (Dako) for 20 minutes at room temperature. Then, it was reacted with Rabbit polyclonal to CD31 (Abcam) diluted 50 times with Dako Real Antibody Diluent (Dako) at room temperature for 30 minutes. After washing with TBS-T solution three times for 5 minutes, the mixture was reacted with Alexa Fluor 488 Goat Anti-rabbit IgG (Molecular probe) diluted 1000 times with Dako Real Antibody Diluent for 30 minutes at room temperature. After washing with a TBS-T solution three times for 5 minutes, the cells were sealed using Vector Shield Mounting Medium with DAPI (Hard Type) (VECTOR). In addition, it replaced with Rabbit polyclonal to CD31 as negative control, and used the section | slice which performed said operation using Negative Control Rabbit Immunoglobulin fraction (Dako). Using the fluorescence microscope (Nikon ECLIPSE TE300) connected with the Super high pressure mercury lamp power supply, the fluorescence image was acquired by the excitation wavelength of 505 nm and the fluorescence wavelength of 520 nm. Based on the obtained image, the amount of fluorescence per unit area was measured by ACT-1C for DXM1200C, and it was found that the amount of fluorescence emitted from the 4T1-luc mSMPD3 KD tumor mass was 58% of the control. From the above results, it was found that tumor blood vessel formation was inhibited by suppressing the expression level of SMPD3 in the tumor.

Example 4 Comparative Analysis of MDA-MB-231 , MCF-7, and MCF-10A MicroRNA Secretion MDA-MB-231 is a cell line isolated from human breast cancer and has lost estrogen sensitivity. It is used as a model for breast cancer with high malignancy and is 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 cells, MCF7 cells, and MCF-10A cells (all purchased from ATCC) are cultured in a medium containing 10% fetal bovine serum (FBS) in RPMI medium, 1 x 10 ⁶ cells After adding 1 fmol of synthetic Cel-miR-39 (miScript miRNA Mimic: manufactured by QIAGEN), microRNA was extracted using RNeasy mini kit (Qiagen) according to the protocol attached to the kit. Next, using the obtained microRNA as a template, TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) and miR-100 TaqMan MicroRNA Assay, miR-29b-1 *
TaqMan MicroRNA Assay, miR-221 * TaqMan MicroRNA Assay, miR-221 TaqMan MicroRNA Assay, miR-146a TaqMan MicroRNA Assay, miR-126 TaqMan MicroRNA Assay, miR-130a TaqMan MicroRNA Assay, miR-222 TaqMan MicroRNA Assay, miR-125b TaqMan MicroRNA Assay, miR-29a TaqMan MicroRNA Assay, miR-720 TaqMan MicroRNA Assay, miR-224 TaqMan MicroRNA Assay, miR-29b TaqMan MicroRNA Assay, miR-1274b TaqMan MicroRNA Assay, miR-1280 TaqMan MicroRNA Assay, miR-210 TaqMan MicroRNA Assay, miR-138 TaqMan MicroRNA Assay, miR-584 TaqMan MicroRNA Assay, miR-140-3p TaqMan MicroRNA Assay, miR-30a TaqMan MicroRNA Assay, miR-151-3p TaqMan MicroRNA Assay, miR-886-3p TaqMan MicroRNA Assay Reverse transcription reaction was performed using miR-593 TaqMan MicroRNA Assay, miR-483-3pTaqMan MicroRNA Assay, and Cel-miR-39 TaqMan MicroRNA Assay (all from Applied Biosystems). That is, microRNA solution 3.3 μL, 100 mM dNTPs (with dTTP) 0.05 μL, MultiScribe Reverse Transcriptase (50 U / μL) 0.33 μL, 10 × Reverse Transcription Buffer 0.5 μL, RNase Inhibitor (20 U / μL) 0.063 μL, Nuclease-free water 1.387 μL and TaqMan MicroRNA Assay (5 ×) 1 μL were 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 3-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) were mixed, and 95 ° C. for 10 minutes. MDA-MB-231 cells of each microRNA, by carrying out PCR reaction using a 7300 Real Time PCR System (Applied Biosystems) by repeating the incubation reaction 40 times after incubation at 95 ° C for 15 seconds and 60 ° C for 1 minute. The amount of secretion in MCF-7 cells and MCF-10A cells was quantified.
As a result, as shown in Table 1, it was found that the secretion levels of these 24 types of microRNAs were significantly increased in MDA-MB-231 cells compared to MCF7 cells and MCF-10A cells.

Example 5 Evaluation of Angiogenesis Inhibitory Activity of MicroRNA 100 μL of pre-miR (Applied Biosystems) and 100 μL OPTI-MEM described in Example 4, mixed solution of 4 μL DharmaFECT1 (Thermo Scientific) and 196 μL OPTI-MEM The mixture was mixed at room temperature and allowed to stand for 30 minutes. Next, after adding the mixture to a 6-well plate, 1 × 10 ⁶ cells suspended in endothelial cell basal medium-2 (Lonza) supplemented with 2 mL of endothelial cell addition factor set-2 (Lonza) MicroRNA transfection was performed by adding normal human umbilical vein endothelial cells (HUVEC cells) (Lonza). On the next day, dispense 120 μL of ECM gel from Engelbreth-Holm-Swarm mouse sarcoma (Sigma) per hole in a 24-well plate and leave it in a CO ₂ incubator for 30 minutes. Solidified. Next, 2 × 10 ⁵ cells / mL of HUVEC cells transfected the day before were dispensed in 500 μL per well. As control cells, cells transfected with NC1 control miRNA (Applied Biosystems) were used. On the next day, in order to evaluate the angiogenic ability, an angiogenesis state of each well was photographed with a microscope (Nikon ECLIPSE TE200) at a magnification of 50 times. Furthermore, the blood vessel length and the number of branch points per certain area in the photograph were measured using image analysis software ImageJ.
Table 2 shows the relative values of the length of blood vessels and the number of branch points formed by cells into which each microRNA has been introduced relative to control cells, and the evaluation results of promotion and suppression are the number of + (promoting activity) and-(suppressing activity). Indicated by A large number of + and-symbols indicates strong activity. The blank represents that neither activity nor inhibition was shown.
As a result, miR-151-3p and miR-886-3p are microRNAs that promote angiogenesis, and miR-29b-1 *, miR-221 *, miR-138, miR-584, and miR are microRNAs that suppress angiogenesis. -30a and miR-146a were identified. miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a and miR-146a have been found to be useful as tumor therapeutic agents because they inhibit angiogenesis. On the other hand, miR-151-3p and miR-886-3p promote angiogenesis, so miR-151-3p inhibitors such as anti-miR-151-3p and miR- such as anti-miR-886-3p 886-3p inhibitors are thought to suppress angiogenesis and have been found to be useful as tumor therapeutic agents.

Example 6 Evaluation of Inhibitory Activity of Anti-mmu-miR-151-3p in Intracellular miR-151-3p In order to confirm the effect of Anti-miR oligonucleotide on miR-151-3p, it is represented by SEQ ID NO: 53 Twelve types of Anti-mmu-miR-151-3p oligonucleotides that are completely complementary to mouse miR-151-3p (mmu-miR-151-3p) and have different chemical modifications Synthesized by Design (Table 3). In the sequences of Table 3, the underlined portion indicates a nucleic acid subjected to LNA modification, and the non-underlined portion indicates a nucleic acid subjected to 2′-OMe modification.

0.01 μg of miR-151-3p sensor vector (SwitchGear Genomics miRNA Synthetic Target), 0.01 μg of mmu-miR-151-3p expression plasmid (treasure bio microRNA Archive Mouse ver.1), and various amounts of various anti-mmu -miR-151-3p was mixed with 20 μL of OPTI-MEM medium containing 0.25 μg of Lipofectamine 2000 and added to a 96-well plate with 5 × 10 ⁴ HEK293 cells. The microRNA sensor vector refers to a plasmid vector in which a sequence complementary to a seed sequence that plays an important role when microRNA binds to a target mRNA is repeatedly inserted into the 3′UTR region of the Kaisei luciferase gene. By introducing a sensor vector for any microRNA into a cell and measuring the luciferase activity of the cell, the activity of the microRNA in the cell can be evaluated. On the next day, firefly luciferase and sea shiitake luciferase activities were measured (Dual-Glo ^™ Luciferase Assay System: Promega). The smaller the value obtained by dividing the sea shiitake luciferase activity by the firefly luciferase activity, the higher the microRNA activity. As a result, intracellular miR-151-3p activity inhibitory effect was observed by applying 3mer or more LNA modification, and intracellular miR-151-3p activity inhibitory effect reached a plateau by applying 4mer or more LNA modification (Figure 3).

Example 7 Stability Evaluation of Anti-mmu-miR-151-3p in Serum After keeping 100 pmol of various anti-mmu-miR-151-3p in a 25% FBS aqueous solution at 37 ° C. for a predetermined period, Analysis of acrylamide gel electrophoresis was performed using the method described above, and the stability was evaluated. Set 15% TBE-Urea Gel 12well (invitrogen CatNo: EC68852BOX) in XCell SureLock Mini-Cell (invitrogen), and 1 × TBE Running Buffer (10 × TBE Running Buffer (invitrogen) diluted with distilled water). Poured. After loading 20 μL (nucleic acid 100 pmol amount) of sample, electrophoresis was performed at 180 V for 90 minutes. Next, the electrophoresed gel was shaken in 0.025% Stains-all solution (sigma) (diluted solution of 25% isopropanol in 4% formamide solution) for 60 minutes. After removing the dye solution and decolorizing with tap water for 1-2 hours, a photograph was taken with LAS2000 (Fuji Film). As a result, it was found that the effect of improving serum stability was observed by applying LNA modification of 3mer or more, and that the effect of improving serum stability reached a plateau by applying LNA modification of 4mer or more (Fig. 4). In addition, anti-miR oligonucleotides (mmu-3mer LNA-1 (corresponding to 3mer-1 in Fig. 4) and mmu-3mer LNA-2 (corresponding to 3mer-2 in Fig. 4), also with 3mer LNA modification) However, mmu-3mer LNA-2 with LNA modification on both 5 'and 3' ends (2mer at 5 'end and 1mer at 3' end) is one end (3mer at 5 'end) The effect of improving serum stability was higher than that of mmu-3mer LNA-1 with LNA modification alone.

Example 8 Evaluation of duplex-forming ability of Anti-mmu-miR-151-3p and mature miR-151-3p 100 pmol of mature miR-151- in the same amount as various anti-mmu-miR-151-3p After mixing 3p, the mixture was incubated at 70 ° C. for 5 minutes, and then returned to room temperature to form anti-mmu-miR-151-3p and miR-151-3p duplexes. Subsequently, acrylamide gel electrophoresis analysis was performed by the method described in Example 7 to separate the double-stranded nucleic acid from the single-stranded nucleic acid, and the double-stranded forming ability was evaluated. As a result, the effect of improving the ability to form a double strand was clearly recognized by applying a 4mer or more LNA modification (FIG. 5).

Example 9 Evaluation of Anti-mmu-miR-151-3p Secretory-type mmu-miR-151-3p Activity Inhibitory Activity 5 μg of mmu-miR-151-3p expression plasmid was transferred to 5 × 10 ⁶ / T- HEK293 cells seeded at a concentration of 75 flasks were transfected using Lipofectamine2000. The next day, the cells were washed with PBS, 15 mL of Advanced-RPMI medium was added, and the culture supernatant was recovered after 2 days. The supernatant was filtered through a 0.2 μm filter to obtain secreted mmu-miR-151-3p.
0.01 μg of miR-151-3p sensor vector was mixed with 20 μL of OPTI-MEM medium containing 0.25 μg of Lipofectamine 2000 and added to a 96-well plate along with 5 × 10 ⁴ HEK293 cells. The next day, after removing the medium, 100 μL of secreted mmu-miR-151-3p obtained by the above-described method was added, and various anti-mmu-miR-151-3p in the amounts shown in FIG. 6 were further added. . The next day, the luciferase activity was measured by the method described in Example 6, and the microRNA activity in each well was evaluated. By applying LNA modification of 3mer or more, a secretory microRNA activity inhibitory effect was observed, and LNA of 4mer or more was observed. It was found that the effect of suppressing the secretory microRNA activity reaches a plateau by applying the modification (FIG. 6).

Example 10 Evaluation of Anti-hsa-miR-151-3p Secretion Type hsa-miR-151-3p Inhibition of Angiogenesis Human miR-151-3p (hsa- comprising the nucleotide sequence represented by SEQ ID NO: 7 Twelve types of Anti-hsa-miR-151-3p oligonucleotides that were completely complementary to miR-151-3p) and differed in chemical modification were synthesized by Gene Design (Table 4). In the sequences of Table 4, the underlined portion indicates a nucleic acid subjected to LNA modification, and the non-underlined portion indicates a nucleic acid subjected to 2′-OMe modification.

1 × 10 ⁶ normal human umbilical vein endothelial cells (HUVEC cells) (Lonza) were suspended in endothelial cell basic medium-2 (Lonza) supplemented with endothelial cell additive factor set-2 (Lonza), The cells were cultured for 1 day in 6-well plates. On the next day, the medium was removed, and instead of the mmu-miR-151-3p expression plasmid, the secreted miR-151-3p prepared by the method described in Example 9 was used using the hsa-miR-151-3p expression plasmid. After 1 mL was added, 100 nmol of various anti-hsa-miR-151-3p was added, and further cultured for 1 day.
120μL of ECM gel from Engelbreth-Holm-Swarm mouse sarcoma (Sigma) melted on ice in a 24-well plate was dispensed per well, and the gel was solidified by standing in a CO ₂ incubator for 30 minutes. . Next, 2 × 10 ⁵ cells / mL of HUVEC cells subjected to the above treatment were dispensed at 500 μL per well. On the next day, in order to evaluate the angiogenic ability, an angiogenesis state of each well was photographed with a microscope (Nikon ECLIPSE TE200) at a magnification of 50 times. Furthermore, the blood vessel length and the number of branch points per certain area in the photograph were measured using image analysis software ImageJ.
Table 3 shows the relative values of the blood vessel length and the number of branch points formed by the cells into which each anti-hsa-miR-151-3p was introduced relative to the control cells in%. As a result, it was found that anti-hsa-miR-151-3p modified with 4mer to 12mer LNA had an inhibitory effect on angiogenic activity by secreted hsa-miR-151-3p (Table 5). In addition, of two anti-miR-151-3p (hsa-3mer LNA-1 and hsa-3mer LNA-2) with 3mer LNA modification, LNA modification at both 5 'and 3' ends In hsa-3mer LNA-2 subjected to the treatment, an angiogenesis inhibitory effect was observed. Inhibition effect was not observed under these test conditions with the other modified anti-miR-151-3p, including all 21mer modified with LNA. Since miR-151-3p is a secreted microRNA derived from cancer cells, the modified Anti-miR-151-3p, which showed an inhibitory effect on angiogenesis in vitro, is a cell secreted from cancer cells. It is considered that it can bind to external miR-151-3p in the same manner and exert an inhibitory effect on angiogenesis even in vivo.

Example 11 Effect on Metastasis Assay in Subcutaneous Transplantation Model of miR-151-3p Hypersecretory Cells Using 1 μg of mmu-miR-151-3p expression plasmid containing mouse miR-151-3p, using LipofectamineLTX kit (Invitrogen) According to the procedure attached to the reagent kit, it was introduced into MDA-MB-231-luc D3H1 cells (Xenogen) seeded at a concentration of 1 × 10 ⁶ cells / well (6-well plate). On the next day, the medium was replaced with 10% FBS / RPMI medium (Invitrogen) containing 2 μg / mL Puromycin (Invitrogen), and the cells were stably cultured for 2 weeks to select stably expressing cells.
In order to measure the amount of mouse miR-151-3p secreted by a plurality of stably expressing cells, 1 fmol of synthetic Cel-miR-39 (miScript miRNA Mimic: manufactured by QIAGEN) was added to 100 μL of the culture supernatant, RNA was extracted using RNeasy mini kit (Qiagen) according to the protocol attached to the kit. Next, reverse transcription using the obtained microRNA as a template using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems), miR-151-3p TaqMan MicroRNA Assay, and Cel-miR-39 TaqMan MicroRNA Assay (Applied Biosystems) Reaction was performed. That is, microRNA solution 3.3 μL, 100 mM dNTPs (with dTTP) 0.05 μL, MultiScribe Reverse Transcriptase (50 U / μL) 0.33 μL, 10 × Reverse Transcription Buffer 0.5 μL, RNase Inhibitor (20 U / μL) 0.063 μL, Nuclease-free water 1.387 μL and TaqMan MicroRNA Assay (5 ×) 1 μL were 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 3-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) were mixed, and 95 ° C. for 10 minutes. After incubation, repeat the incubation cycle of 95 ° C for 15 seconds and 60 ° C for 1 minute 40 times using the 7300 Real Time PCR System (Applied Biosystems). The amount of secretion was quantified. The stable expression cell line (miR-151-3p hypersecretory cell) having the highest calculated value was used in the following experiment.
Five 7-week-old male Balb / cathymic nude mice were transplanted with 1 × 10 ⁶ miR-151-3p hypersecreting cells suspended in PBS subcutaneously in both inguinal regions. As a control group, Balb / cathic nude mice transplanted with 1 × 10 ⁶ MDA-MB-231-luc D3H1 cells were used. 14 days after transplantation, when measuring luciferase activity in the vicinity of lymph nodes using in vivo imaging method (IVIS Xenogen), lymph node metastasis of mice transplanted with miR-151-3p hypersecretory cells was compared with the control group, It was found that there was a significant increase (FIG. 7).

Example 12 Effect of miR-151-3p oversecretory cells on tumor angiogenesis in a subcutaneous transplantation model A tumor mass in the inguinal region of Balb / cathic nude mice 20 days after miR-151-3p oversecretory cell transplantation was removed Formalin fixation. After that, 5 μm paraffin-embedded sections were immunostained with the antibody against CD31, a vascular endothelium-specific protein (Rabbit polyclonal to CD31 ab28364 (Abcam)), according to the following procedure, and the ability to form tumor blood vessels was confirmed. evaluated. As a control group, a tumor mass at the groin part of Balb / cathic nude mice transplanted with 4T1-luc cells was used. First, the section was washed with xylene three times for 5 minutes, and then washed for 5 minutes in the order of 99.5% ethanol, 80% ethanol, and 70% ethanol. Further, after washing 3 times with distilled water for 5 minutes, it was immersed in running water for 10 minutes. Subsequently, after washing 3 times with distilled water for 5 minutes, autoclaving was performed at 121 ° C. for 5 minutes in 0.5 M Tris buffer (pH = 10). Then, after cooling by standing at room temperature for 30 minutes, washed with distilled water for 5 minutes, 3 times, followed by TBS-T solution (0.05M Tris, 0.3M NaCl, 0.1% Tween20 aqueous solution) for 5 minutes. Washed twice. After surrounding the tissue with a super pap pen, it was immersed in Image-iT Ex Signal Enhancer (Molecular probe) at room temperature for 30 minutes. After washing with TBS-T solution, it was immersed in Dako Protein Block (Dako) for 20 minutes at room temperature. Then, it was reacted with Rabbit polyclonal to CD31 (Abcam) diluted 50 times with Dako Real Antibody Diluent (Dako) at room temperature for 30 minutes. After washing with TBS-T solution three times for 5 minutes, the mixture was reacted with Alexa Fluor 488 Goat Anti-rabbit IgG (Molecular probe) diluted 1000 times with Dako Real Antibody Diluent for 30 minutes at room temperature. After washing with a TBS-T solution three times for 5 minutes, the cells were sealed using Vector Shield Mounting Medium with DAPI (Hard Type) (VECTOR). In addition, it replaced with Rabbit polyclonal to CD31 as negative control, and used the section | slice which performed said operation using Negative Control Rabbit Immunoglobulin fraction (Dako). Using the fluorescence microscope (Nikon ECLIPSE TE300) connected with the Super high pressure mercury lamp power supply, the fluorescence image was acquired by the excitation wavelength of 505 nm and the fluorescence wavelength of 520 nm. When the amount of fluorescence per unit area was measured by ACT-1C for DXM1200C, it was found that the amount of fluorescence emitted from the miR-151-3p hypersecretory cell tumor mass was 147% of the control. From the above results, it was found that the formation of tumor blood vessels was promoted by increasing the amount of miR-151-3p secretion (FIG. 8).

Example 13 Mechanism analysis of angiogenesis-promoting action of miR-151-3p When hsa-miR-151-3p was transfected and an in vitro angiogenesis assay was performed by the method described in Example 5, VEGF receptor 2 When KI 8751 (Tocris Bioscience), a phosphorylation inhibitor, was added at a concentration of 10 nM, it was found that the effect of promoting angiogenesis by hsa-miR-151-3p disappeared (Table 6). This suggests that miR-151-3 exerts an angiogenesis promoting action by regulating the VEGF signal pathway.

Example 14 Suppression of DLL4 signal by miR-151-3p Whether or not the DLL4 signal pathway is blocked by hsa-miR-151-3p was examined by the Western analysis method described below, hsa-miR-151- Suppression of DLL4 expression in HUVEC cells was observed by 3p transfection (FIG. 9). A lysate of 10 μg of HUVEC cells per well was electrophoresed at 200 V for 1 hour using polyacrylamide gel (Supersep Ace 10% 13 well Wako Pure Chemical Industries). The obtained acrylamide gel was transferred to a PVDF membrane at 20 V for 30 minutes using a semi-driving system. Next, after blocking the PVDF membrane with a 3% skim milk / T-TBS solution at room temperature for 1 hour, the antibody diluted appropriately (anti-hDLL4 antibody (DLL4 Antibody (C-term): ABGENT, AP9964a) 1 / 1,000 dilution, Anti-hactin antibody (Anti-Actin, clone C4: MILLIPORE, MAB1501) was treated with 1 / 10,000 dilution for 1 hour at room temperature. After washing with T-TBS solution for 10 minutes three times, the mixture was incubated with a secondary antibody (HRP-conjugated anti-mouse IgG antibody or HRP-conjugated anti-rabbit IgG antibody) diluted 1 / 3,000. Furthermore, after washing with a T-TBS solution for 10 minutes three times, a protein bound to an antibody was detected using a reagent for measuring HRP activity (Applied Biosystems) (LAS2000 Fuji Film).

Example 15 Identification of miR-151-3p Target Genes Among miR-151-3p target gene groups predicted based on a target gene prediction program for microRNAs Targetscan (http://www.targetscan.org/) We tried to extract genes related to angiogenesis. As a result, it was found that a sequence complementary to the seed sequence of miR-151-3p exists on the 3 ′ UTR of the Akt3 gene constituting the VEGF signaling pathway. Therefore, whether or not has-miR-151-3p can act on the 3′UTR of hAkt3 was determined using the hAkt3 3′UTR vector (miTarget miRNA 3′UTR Target Sequence Expression Clone: GeneCopia) as described in Example 6. A luciferase assay was performed to investigate. As a result, miR-151-3p was found to suppress the luciferase activity of HEK293 cells into which the hAkt3 3′UTR vector was introduced in a concentration-dependent manner (FIG. 10).
Next, the endogenous hAkt3 protein level when hsa-miR-151-3p was introduced into HUVEC cells was examined by the Western blotting method described below. A lysate of 10 μg of HUVEC cells per well was electrophoresed at 200 V for 1 hour using polyacrylamide gel (Supersep Ace 10% 13 well Wako Pure Chemical Industries). The obtained acrylamide gel was transferred to a PVDF membrane at 20 V for 30 minutes using a semi-driving system. Next, the PVDF membrane was blocked with a 3% skim milk / T-TBS solution at room temperature for 1 hour, and then diluted appropriately (anti-hAkt3 antibody (Anti-Akt3 / PKBγ, clone GMA104: MILLIPORE, 05-780) The solution was treated with an anti-hactin antibody (Anti-Actin, clone C4: MILLIPORE, MAB1501) at 1,000 dilution for 1 hour at room temperature. After washing with T-TBS solution for 10 minutes three times, the mixture was incubated with a secondary antibody (HRP-conjugated anti-mouse IgG antibody or HRP-conjugated anti-rabbit IgG antibody) diluted 1 / 3,000. Furthermore, after washing with a T-TBS solution for 10 minutes three times, a protein bound to an antibody was detected using a reagent for measuring HRP activity (Applied Biosystems) (LAS2000 Fuji Film). As a result, as shown in FIG. 11, it was found that 10 nM miR-151-3p decreased hAkt3 protein by about half. From the above study, it was found that Akt3 is one of the target genes of miR-151-3p.
When the Akt3 gene was knocked down using siRNA (AKT3 HSS115177 invitrogen) in the presence of VEGF, it was found that the increased expression of DLL4 was canceled (FIG. 12). Furthermore, administration of Akt3 siRNA and DLL4 antibody (DLL4 antibody (C-term) ABGENT) increased the number of blood vessels formed (Table 7). It was confirmed that it occurred.
These results suggest that miR-151-3p blocks the expression of Akt3 and DLL4, thereby blocking VEGF feedback signals and leading to abnormal angiogenesis.

The tumor therapeutic agent of the present invention suppresses tumor angiogenesis and is useful for tumor metastasis and prevention, particularly for breast cancer treatment and prevention. Further, malignant cancer, particularly malignant breast cancer can be determined by the cancer determination method of the present invention. Furthermore, the present invention can also provide an agent for determining malignant cancer and a method for screening a substance having an action of suppressing tumor angiogenesis.

This application is based on Japanese Patent Application No. 2011-047647 filed in Japan (filing date: March 4, 2011), the contents of which are incorporated in full herein.

Claims

The following (a) inhibitor or tumor therapeutic agent containing the nucleic acid according to (b):
(A) an miR-151-3p inhibitor, an miR-886-3p inhibitor, or an SMPD3 inhibitor,
(B) (1) a nucleotide sequence represented by any of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, or SEQ ID NOs: 1-6 A nucleic acid comprising a nucleotide sequence having a nucleotide sequence having 70% or more identity and having a target gene expression suppressing activity, or (2) a nucleic acid that is a vector capable of expressing the nucleic acid of (1) above.
The agent according to claim 1, wherein the miR-151-3p inhibitor is the nucleic acid according to (1) or (2) below:
(1) a nucleic acid comprising a nucleotide sequence comprising 70% or more of the nucleotide sequence represented by SEQ ID NO: 9, and comprising a nucleotide having an activity of suppressing the function of miR-151-3p,
(2) A nucleic acid that is an expression vector capable of expressing the nucleic acid of (1) above.
The agent according to claim 1, wherein the miR-886-3p inhibitor is the nucleic acid according to the following (1) or (2):
(1) a nucleic acid comprising a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, and comprising a nucleotide having an activity of suppressing the function of miR-886-3p,
(2) A nucleic acid that is an expression vector capable of expressing the nucleic acid of (1) above.
The agent according to claim 1, wherein the SMPD3 inhibitor is a substance selected from the group consisting of the following (1) to (5):
(1) an antisense nucleic acid against a transcription product of a gene encoding SMPD3,
(2) a ribozyme nucleic acid for the transcription product of the gene encoding SMPD3,
(3) a nucleic acid having RNAi activity for a transcription product of a gene encoding SMPD3 or a precursor thereof,
(4) an antibody that binds to SMPD3,
(5) A low molecular weight compound that binds to SMPD3.
The agent according to any one of claims 1 to 4, wherein the nucleic acid is single-stranded or double-stranded.
The agent according to any one of claims 1 to 3, wherein the nucleic acid is RNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6, 9, and 10, or RNA comprising a partial sequence thereof, or a modified form thereof.
The agent according to any one of claims 1 to 3 and 6, wherein the nucleic acid is RNA consisting of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 6, 9, and 10, or a modified product thereof.
The agent according to claim 2, wherein the nucleic acid is a single-stranded RNA comprising the nucleotide sequence represented by SEQ ID NO: 9 or SEQ ID NO: 54 or a modified product thereof.
The agent according to claim 8, wherein the nucleic acid is at least one single-stranded RNA containing a modified nucleotide of either 2'-OMe modified nucleotide or LNA modified nucleotide.
The agent according to claim 9, wherein the single-stranded RNA comprises 3 or more LNA-modified nucleotides.
The agent according to claim 9, wherein the single-stranded RNA comprises 4 or more LNA-modified nucleotides.
The agent according to claim 9, wherein the single-stranded RNA comprises 3 to 12 LNA-modified nucleotides.
The agent according to claim 9, wherein the single-stranded RNA comprises 4 to 12 LNA-modified nucleotides.
The agent according to claims 8 to 13, wherein the single-stranded RNA comprises at least one LNA-modified nucleotide at both the 5 'end and the 3' end.
The agent according to any one of claims 8 to 14, wherein all nucleotides are single-stranded RNAs consisting of modified nucleotides of either 2'-OMe modified nucleotides or LNA modified nucleotides.
The agent according to claim 10, wherein the single-stranded RNA is a single-stranded RNA consisting of a nucleotide sequence represented by any one selected from SEQ ID NOs: 30 to 38 and 43 to 50.
The agent according to claim 10, wherein the single-stranded RNA is a single-stranded RNA consisting of a nucleotide sequence represented by any one selected from SEQ ID NOs: 43 to 48 and 50.
The agent according to any one of claims 1 to 17, which inhibits angiogenesis.
The agent according to any one of claims 1 to 17, which inhibits tumor angiogenesis.
The agent according to any one of claims 1 to 17, which suppresses tumor metastasis.
The agent according to any one of claims 1 to 20, wherein the tumor is breast cancer.
The inhibitor or nucleic acid according to any one of claims 3 and 8 to 17, which suppresses angiogenesis abnormality caused by overexpression of miR-151-3p.
The inhibitor or nucleic acid according to any one of claims 1 to 17, for treating a tumor.
The inhibitor or nucleic acid according to any one of claims 1 to 17, which inhibits angiogenesis.
The inhibitor or nucleic acid according to any one of claims 1 to 17, which inhibits tumor angiogenesis.
The inhibitor or nucleic acid according to any one of claims 1 to 17, which suppresses tumor metastasis.
Use of the inhibitor or nucleic acid according to any one of claims 1 to 17 for producing a tumor therapeutic agent.
Use of the inhibitor or nucleic acid according to any one of claims 1 to 17 for producing an angiogenesis inhibitor.
Use of the inhibitor or nucleic acid according to any one of claims 1 to 17 for producing a tumor angiogenesis inhibitor.
Use of the inhibitor or nucleic acid according to any one of claims 1 to 17 for producing a tumor metastasis inhibitor.
A method for treating a tumor in a human, comprising administering the inhibitor or nucleic acid according to any one of claims 1 to 17 to the human.
A method for inhibiting angiogenesis in a human comprising administering the inhibitor or nucleic acid according to any one of claims 1 to 17 to the human.
A method for inhibiting tumor angiogenesis in a human comprising administering the inhibitor or nucleic acid according to any one of claims 1 to 17 to the human.
A method for suppressing tumor metastasis in a human comprising administering the inhibitor or nucleic acid according to any one of claims 1 to 17 to the human.
MiR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR- 221, miR-126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR-140- Based on measuring the secretion level or concentration of 3p, miR-593 or miR-483-3p and positive correlation between the secretion level or the concentration and malignant cancer, A method for determining cancer, comprising determining.
36. The method according to claim 35, wherein the cancer is breast cancer.
miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a, miR-146a, miR-151-3p, miR-886-3p, miR-100, miR-221, miR- 126, miR-130a, miR-222, miR-125b, miR-29a, miR-720, miR-224, miR-29b, miR-1274b, miR-1280, miR-210, miR-140-3p, miR- An agent for determining malignant cancer, comprising a nucleic acid probe capable of specifically detecting 593 or miR-483-3p.
The agent according to claim 37, wherein the cancer is breast cancer.
A method for searching for a substance capable of suppressing tumor angiogenesis, comprising the following steps:
(1) contacting a test substance with a cell capable of measuring secretion or expression of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a;
(2) measuring the amount of miR-29b-1 *, miR-221 *, miR-138, miR-584, miR-30a or miR-146a secreted or expressed in cells contacted with the test substance, Comparing the amount of secretion or expression with the amount of secretion or expression in control cells not contacted with the test substance; and (3) based on the comparison result of (2) above, miR-29b-1 *, miR-221 * Select a test substance that up-regulates the secretion or expression level of miR-138, miR-584, miR-30a, or miR-146a as a substance that can suppress tumor angiogenesis.
A method for searching for a substance capable of suppressing tumor angiogenesis, comprising the following steps:
(1) contacting a test substance with a cell capable of measuring the secretion, expression or function of miR-151-3p, miR-886-3p or SMPD3;
(2) Measure the secretion amount, expression level or function of miR-151-3p, miR-886-3p or SMPD3 in cells contacted with the test substance, and determine the secretion amount, expression level or function of the test substance Comparing with the secreted amount, expression level or function in the non-contacted control cells; and (3) secreted amount, expression of miR-151-3p, miR-886-3p or SMPD3 based on the comparison result of (2) above A test substance that down-regulates the amount or function is selected as a substance capable of suppressing tumor angiogenesis.
It consists of a nucleotide sequence represented by SEQ ID NO: 9 or SEQ ID NO: 54, all nucleotides are composed of either 2′-OMe modified nucleotides or LNA modified nucleotides, and 3 to 12 LNA modified nucleotides Including single-stranded RNA.
42. The single stranded RNA of claim 41, wherein the single stranded RNA comprises 4 to 12 LNA modified nucleotides.
43. The single-stranded RNA according to claim 41 or 42, wherein the single-stranded RNA comprises at least one LNA-modified nucleotide at both the 5 'end and the 3' end.
The single-stranded RNA comprising a nucleotide sequence represented by any one selected from SEQ ID NOs: 30 to 38 and 43 to 50.