Composition for diagnosis, prevention or treatment of vascular smooth muscle cell proliferative disorders using MIRNA inhibitors

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a vascular smooth muscle cell proliferative disease, comprising an miRNA (microRNA) inhibitor as an active ingredient; a kit for diagnosing a vascular smooth muscle cell proliferative disease, comprising a preparation capable of detecting miRNA; a method for providing information on the diagnosis of the disease; and a treatment method. The present invention has discovered a subset of miRNAs involved in a proliferative disease of smooth muscle cells, miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p, and the expression of these miRNAs is significantly increased by more than 5 times in damaged arteries compared to the control group. In particular, miR-370-3p has been confirmed to be highly expressed in the tubular tissues of arteriosclerosis patients, making it useful for diagnosing diseases related thereto, and can also be used for treating a vascular smooth muscle cell proliferative disease using the same.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating a vascular smooth muscle cell proliferative disease, which contains an miRNA (microRNA) inhibitor as an active ingredient; a kit for diagnosing a vascular smooth muscle cell proliferative disease, which contains a preparation capable of detecting miRNA; a method for providing information on the diagnosis of the disease; and a treatment method. [Background technology]

[0002] Normal arterial vessels are composed of a monolayer of healthy endothelial cells (EC) and two major cell types: contractile smooth muscle cells (SMCs). However, internal accumulation of low-density lipoproteins combined with impaired blood flow can cause inflammation in ECs, and monocytes / macrophages attach to the inflamed EC lesions, infiltrate the EC monolayer, and absorb lipid particles to become foam cells. The generated macrophage-driven foam cells and immune cells in the atherosclerotic lesions secrete a number of growth factors and cytokines that stimulate smooth muscle cells to reverse differentiation and form a fibrous cap. The entire atherogenic process, including the initial inflammatory response, eventually leads to the occlusion of the arterial lumen. Currently, the only way to restore blocked arteries in patients with atherosclerosis is to perform angioplasty and stent surgery at the same time, but after surgery, endothelial damage leads to thrombosis, a major complication, which induces additional neointima proliferation, known as in-stent restenosis. The process of such restenosis complications is again similar to the proliferation and migration of reverse-differentiated smooth muscle cells in the later stages of arteriosclerosis, thus suggesting that any targeted approach may not be the correct solution for preventing arteriosclerosis.

[0003] After their discovery in 1993 while studying the lin-4 gene in C. elegans, the function of miRNAs as translation regulators has attracted much interest in the past decades. A unique feature of miRNAs is that they simultaneously regulate the expression of various genes in their 3'UTRs, and indeed, various studies have attempted to find and characterize vascular miRNAs that regulate the growth of vascular smooth muscle cells (Farina, 2020 #2619; Torella, 2018 #2618; Ji, 2007 #2617). Nevertheless, there has been no genome-wide screening of common vascular miRNAs in rodents and humans. Summary of the Invention [Problem to be solved by the invention]

[0004] Under these circumstances, the present inventors have made intensive research to screen vascular miRNAs associated with hyperproliferation and migration of arterial smooth muscle cells, and as a result, using a standard animal model showing smooth muscle cell proliferation, identified four important miRNAs that regulate the proliferation and migration of arterial smooth muscle cells. In particular, they confirmed that miR-370-3p is a new miRNA associated with a proliferative disease of smooth muscle cells that is very important in atherosclerosis, thereby completing the present invention. [Means for solving the problem]

[0005] One object of the present invention is to provide a pharmaceutical composition for preventing or treating a proliferative disorder of vascular smooth muscle cells, comprising an miRNA (microRNA) inhibitor as an active ingredient. Another object of the present invention is to provide a diagnostic kit for vascular smooth muscle cell proliferative disorders, comprising a preparation capable of detecting miRNA.

[0006] Another object of the present invention is to provide a method for providing information for the diagnosis of vascular smooth muscle cell proliferative disorders. Another object of the present invention is to provide a method for treating a vascular smooth muscle cell proliferative disorder, comprising administering the pharmaceutical composition containing an miRNA expression inhibitor to an individual, other than a human, in need thereof. Effect of the Invention

[0007] The present invention has discovered a subset of miRNAs involved in proliferative diseases of vascular smooth muscle cells, namely, miR-132-3p, miR-370-3p, miR-130b-5p and miR-410-3p. The expression of these miRNAs is significantly increased by more than five-fold in damaged arteries compared to control groups. In particular, miR-370-3p has been confirmed to be highly expressed in the tubular tissues of arteriosclerosis patients, making it useful for diagnosing the above diseases and can also be used to prevent or treat proliferative diseases of vascular smooth muscle cells. [Brief description of the drawings]

[0008] [Figure 1a] Heatmap analysis of differentially expressed miRNAs 3 and 5 days after arterial injury compared to sham control from a balloon-injured arterial animal model. [Figure 1b] Graph showing 8 upregulated and 2 downregulated miRNAs that showed 5-fold or greater change in expression levels among differentially expressed miRNAs. [Figure 1c] FIG. 1B is a graph confirming that of the eight upregulated miRNAs in FIG. 1B, six were significantly expressed in human smooth muscle cells. [Figure 2a] In order to confirm the role of upregulated miRNAs, human vascular smooth muscle cells transfected with miRNA inhibitors were used. The results showed that miR-132-3p and miR-370-3p showed significant inhibitory effects on smooth muscle cell proliferation, and miR-130b-5p, miR-132-3p, and miR-410-3p significantly inhibited monocyte adhesion. [Figure 2b]1 is a graph showing that miR-132-3p and miR-370-3p inhibitors induce cell cycle arrest at the G1 phase and inhibit the time-dependent proliferation of human vascular smooth muscle cells. [Figure 2c] 1 is a graph showing that miR-132-3p and miR-370-3p inhibitors induce cell cycle arrest at the G1 phase and inhibit the time-dependent proliferation of human vascular smooth muscle cells. [Figure 2d] 1 is a graph showing that miR-132-3p and miR-370-3p inhibitors significantly increase the levels of cyclin-dependent kinase inhibitors p21 and p27. [Figure 2e] This is a graph confirming that miR-132-3p is involved in smooth muscle cell phenotype transition, as miR-132-3p inhibitors restored SMA levels, thereby converting smooth muscle cells to a contractile phenotype. [Figure 2f] This is a graph which reaffirms that the synthetic phenotype of human vascular smooth muscle cells was transformed into a contractile phenotype by the miR-132-3p inhibitor through F-actin filaments. [Figure 3a] The arterial expression levels of the four selected miRNAs were confirmed through in situ hybridization, and it was found that miR-132-3p and miR-370-3p were significantly increased in balloon-injured arteries, and miR-130b-5p and miR-410-3p were induced by balloon injury. [Figure 3b] 1 shows photographs and graphs confirming that catheter-mediated local intramural delivery of four selected miRNA inhibitors significantly regresses neointimal proliferation in balloon-injured lesions compared to the control group. [Figure 3c] Among miRNAs, inhibitors of miR-130b-5p exhibited antiproliferative efficacy in ECs by promoting the recovery of the EC monolayer through re-endothelialization. [Figure 4a]We profiled differentially expressed genes (DEGs) in injured arteries. [Figure 4b] Up- and down-regulated DEGs were identified through heatmap analysis. [Figure 4c] 1 is a graph showing that predicted target genes of the selected 4 miRNAs were down-regulated in the balloon-injured carotid artery compared to the sham control group through real time PCR. [Figure 4d] This is a graph confirming through real time PCR that the target genes of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p miRNAs are SOCS2, BMP7, TSPAN2, and SMAD6, respectively. [Figure 5a] High expression of miR-370-3p was confirmed in tubular tissue sections from human patients with arteriosclerosis type II and type IV lesions. [Figure 5b] We investigated the relationship between miR-370-3p and BMP7 through serum stimulation, which induced miR-370-3p expression but reduced BMP7 expression. [Figure 5c] It was confirmed that BMP7 protein levels were increased in vascular smooth muscle cells through the inhibition of miR-370-3p. [Figure 5d] It was confirmed that miR-370-3p mimics reduced luciferase expression containing the wild-type UTR but not the negative control group in which two consecutive nucleotides within the miR-370-3p target region of human BMP7-3'UTR were mutated. [Figure 5e] This confirms the induction of SMAD1 / 5 / 9 phosphorylation in vascular smooth muscle cells by BMP7 treatment. [Figure 5f] This demonstrated that reduction of BMP7 by siRNA transfection restored smooth muscle cell proliferation suppressed by miR-370-3p inhibitor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] This will be described in detail as follows. However, the scope of the present invention is not limited to the following specific description. In other words, each description and embodiment disclosed in the present invention can be applied to each different description and embodiment, and all combinations of various elements disclosed in the present invention belong to the scope of the present invention.

[0010] In order to achieve the above-mentioned object, as one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a vascular smooth muscle cell proliferative disorder, comprising an miRNA (microRNA) inhibitor as an active ingredient.

[0011] The present invention also provides a use of an miRNA (microRNA) inhibitor for the prevention or treatment of a vascular smooth muscle cell proliferative disorder. In the present application, the term "miRNA" may be written as microRNA, microRNA, etc., and is a small RNA that plays a role in controlling gene expression in an organism, specifically, a small RNA containing 20 to 25 nucleotides that plays an important regulatory role in the gene expression process through the inhibition of target mRNA translation by a complementary base pair sequence with the target mRNA 3'UTR (untranslated region). The miRNA plays an important role in cellular functions including proliferation, differentiation, apoptosis, etc., and is an evolutionarily conserved regulator present in all animals, and some miRNAs can regulate gene expression through epigenetic regulatory mechanisms (histone modification, DNA methylation, etc.) associated with their promoter sites.

[0012] The miRNAs used in the present invention regulate the hyperproliferation, phenotype transition, migration, etc. of smooth muscle cells, and specifically, miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p were identified through a balloon-injured artery model. Furthermore, their core target genes were further confirmed, and in particular, miR-370-3p was discovered to be a new miRNA associated with atherosclerosis, and was first identified by the present inventors.

[0013] The term "miRNA inhibitor" as used herein means a preparation that reduces the expression or activity of each miRNA in cells, and means blocking miRNA that acts directly on each miRNA or indirectly on upper regulators to regulate the expression of target genes. In the present invention, the miRNA inhibitor means regulating vascular smooth muscle cell function and in vivo neointimal proliferation by suppressing miRNAs up-expressed in injured arteries, specifically, but not limited to, each inhibitor of miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p.

[0014] In the present invention, the miRNA suppression can be performed using an inhibitor specific to each of miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p, and specifically, can be an siRNA aptamer, antisense oligonucleotide, ribozyme, or compound specific to each miRNA, but is not limited thereto. For the purpose of the present invention, the compound can be any compound that suppresses miRNA associated with in vivo neointimal proliferation.

[0015] For the purpose of the present invention, the injured artery was artificially injured in order to screen for vascular miRNAs whose expression is associated with hyperproliferation of arterial smooth muscle cells. Specifically, a model in which a rat carotid artery was injured using a balloon was prepared and used in the experiment.

[0016] In one embodiment of the present invention, we identified miRNAs that were differentially expressed in injured arteries. Among the 62 miRNAs that were differentially expressed on days 3 and 5 after injury compared to the control group, we confirmed their expression in human aortic smooth muscle cells, and finally identified six upregulated miRNAs (Figure 1).

[0017] The term "upregulated" refers to an increase in mature miRNAs due to increased transcription and processing of specific miRNAs, and in the present invention, refers to increased biosynthesis in an injured artery model compared to a control. For purposes of the present invention, increased expression specifically refers to a significant increase of 5-fold or more compared to a control, but is not limited thereto.

[0018] In this application, the term "vascular smooth muscle cells" refers to cells that constitute the inner wall of blood vessels and function to maintain a constant blood pressure through contraction and relaxation. In normal blood vessels, smooth muscle cells differentiate to produce proteins necessary for contraction and relaxation and stop cell proliferation, but if a problem occurs in the function and they undergo retrodifferentiation, the blood vessels become narrower, causing diseases such as arteriosclerosis and vascular restenosis. Such vascular diseases are closely related to the function of vascular smooth muscle cells, and therefore vascular smooth muscle cell models are the main cell models in vascular disease research.

[0019] In this application, the term "proliferation" refers to an increase in the number of cells in the body of a multicellular organism, usually by cell division of an organism or tissue cell, etc. In the present invention, the cell proliferation refers to the proliferation of vascular smooth muscle cells, and such excessive proliferation of vascular smooth muscle cells is an important factor in the progression of arteriosclerosis lesions.

[0020] In the present application, the term "vascular smooth muscle cell proliferative disease" refers to a disease caused by excessive proliferation of vascular smooth muscle cells. The vascular smooth muscle cell proliferative disease includes not only vascular stenosis, vascular restenosis, atherosclerosis, and the like, which are directly caused by proliferation of vascular smooth muscle cells, but also cardiovascular diseases such as heart failure, myocardial infarction, angina pectoris, arrhythmia, hypertensive heart disease, congenital heart disease, stroke, and peripheral vascular stenosis, which are secondarily induced or aggravated by these diseases.

[0021] In the present invention, the vascular smooth muscle cell proliferative disease may specifically be vascular stenosis, vascular restenosis, atherosclerosis, or the like. The vascular stenosis is a disease in which the inside of blood vessels becomes abnormally narrowed due to inflammation, blood clots, and excessive proliferation of smooth muscle cells after the vascular wall is damaged, resulting in a decrease in blood flow. Vascular restenosis is the recurrence of vascular stenosis, and is often caused after vascular surgery, such as expanding the vascular lumen or reopening blocked blood vessels to resolve the narrowing of blood vessels. Vascular surgery, such as angioplasty, such as stent / balloon angioplasty, and vascular bypass or vascular graft surgery, such as coronary artery bypass surgery, damage the blood vessels themselves or cause inflammation, resulting in vascular stenosis. Atherosclerosis is a disease in which fat is deposited or fibrotic in the inner layer of the artery, and vascular restenosis that occurs after stent insertion due to the progression of arteriosclerosis and vasodilation is known to be caused by the proliferation, migration, and extracellular matrix secretion of vascular smooth muscle cells.

[0022] As used herein, the term "prevention" refers to any action of suppressing or delaying the onset of a vascular smooth muscle cell proliferative disease by administering the pharmaceutical composition according to the present invention, and "treatment" refers to any action of improving or beneficially modifying the symptoms of an individual suspected of or suffering from a vascular smooth muscle cell proliferative disease by administering the pharmaceutical composition.

[0023] The pharmaceutical composition of the present invention can prevent or treat diseases associated with miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p by inhibiting the activity and / or expression of the same.

[0024] The pharmaceutical composition according to the present invention may contain, as an active ingredient, a miR-132-3p, miR-370-3p, miR-130b-5p or miR-410-3p inhibitor in an amount of 0.1 to 75% by weight, more preferably 1 to 50% by weight, based on the total weight of the composition.

[0025] In addition, the pharmaceutical composition may further include a complex with various nucleic acid carriers (viral or non-viral carriers) known in the art to increase the efficiency of in vivo delivery of the miRNA inhibitor. As the viral carriers, specifically, lentivirus, adenovirus, and adeno-associated virus are used to deliver a vector encoding miRNA to the nucleus of a cell and express the miRNA inhibitor. As the non-viral carriers, inorganic nanoparticle-based carriers that can adjust the size and shape using inorganic materials such as gold, carbon, and silica, polymer-based carriers such as PLGA and PEI that are particularly deformed for effective delivery to specific cells, and lipid carriers using liposomes that are formed of a lipid bilayer and have water as the internal phase, and can encapsulate and deliver nucleic acids, etc., can protect the miRNA inhibitor and improve its stability during blood circulation.

[0026] The pharmaceutical composition can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can be administered singly or multiple times. It is important to administer an amount that provides maximum efficacy with minimal side effects while taking into account all of the above factors, and this can be easily determined by those skilled in the art.

[0027] In addition, the pharmaceutical composition can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dosage varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the administration route and time, but can be appropriately selected by those skilled in the art.

[0028] In one embodiment of the present invention, upregulated miRNAs that regulate vascular smooth muscle cell function in vitro were identified, and it was found that miR-132-3p and miR-370-3p showed significant inhibitory effects on vascular smooth muscle cell proliferation, and that miR-130b-5p, miR-132-3p, and miR-410-3p significantly inhibited monocyte adhesion to vascular smooth muscle cells. Through in-depth investigation of proliferation-related miR-132-3p and miR-370-3p, it was confirmed that they may regulate phenotype transition and proliferation signals, respectively (Figure 2).

[0029] In addition, in one embodiment of the present invention, in order to identify upregulated miRNAs that regulate neointimal proliferation in vivo, the arterial expression levels of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p were investigated. As a result, it was found that the expression of miR-132-3p and miR-370-3p was significantly increased in the balloon-injured carotid artery compared to the sham control group, miR-130b-5p and miR-410-3p were also substantially induced by balloon injury, and inhibitors of the above miRNAs significantly regressed neointimal proliferation in the balloon-injured lesion compared to the control group, and miR-130b-5p inhibitors promoted the recovery of the monolayer, called reendothelialization, thereby confirming that these four miRNAs play distinct roles in arterial homeostasis, such as endovascular inflammation and proliferation (Figure 3).

[0030] In addition, in one embodiment of the present invention, to identify the core target genes of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p, differentially expressed genes (DEGs) were used in the injured artery at two time points, and DEGs predicted to be common to rats and humans through a target prediction database were selected as target genes of each miRNA. It was confirmed that these target genes were down-regulated in the injured artery, and it was revealed that the core target genes of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p were Suppressor of Cytokine Signalling 2 (SOCS2), Bone morphogenetic protein 7 (BMP7), Tetraspanin-2 (TSPAN2), and SMAD family member 6 (SMAD6) (FIG. 4).

[0031] In addition, in one embodiment of the present invention, miR-370 was detected in the blood of patients with unstable angina, type 2 diabetes, and hyperlipidemia, and the biological importance of the miR-370 / bone morphogenic protein (BMP)-7 axis in smooth muscle cell proliferation was investigated. As a result, high expression of miR-370-3p was confirmed in tubular tissue sections of human patients with arteriosclerosis type II and type IV lesions. Therefore, it was confirmed that miR-370 is a new miRNA related to atherosclerosis, and that the level of BMP7 protein was increased in human vascular smooth muscle cells through miR-370 inhibition, BMP7 treatment significantly induced SMAD1 / 5 / 9 phosphorylation in human smooth muscle cells, and depletion of BMP7 prevented the inhibition of smooth muscle cell proliferation by miR-370 inhibitors, confirming that miR-370-dependent BMP7 expression is a prerequisite for smooth muscle cell growth and neointimal proliferation occurring in injured arteries (Figure 5).

[0032] Therefore, based on the cell proliferation inhibitory effects of miR-132-3p, miR-370-3p, miR-130b-5p and miR-410-3p of the present invention, it can be expected that the incorporation of the miRNA inhibitors in vascular smooth muscle cells can be used to prevent or treat vascular smooth muscle cell proliferative diseases.

[0033] In order to achieve the above-mentioned object, in another embodiment of the present invention, the present invention provides a diagnostic kit for vascular smooth muscle cell proliferative disorders, comprising a preparation capable of detecting miRNA. The "miRNA" and "vascular smooth muscle cell proliferative disorder" are as described above.

[0034] In this application, the term "diagnosis" means to confirm the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis can be interpreted as an act of objectively determining whether or not there is hyperproliferation or migration of vascular smooth muscle cells in a patient of interest, in relation to a vascular smooth muscle cell proliferative disorder.

[0035] The diagnostic kit of the present invention is used for qualitatively and / or quantitatively detecting miR-132-3p, miR-370-3p, miR-130b-5p and miR-410-3p or their target genes in a sample.

[0036] Specifically, the kit may be selected from the group consisting of a microarray, an aptamer chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a blotting kit, an immunoprecipitation kit, an immunofluorescence test kit, a protein chip kit, an RT-PCR kit, and a combination thereof, and more specifically, may be an RT-PCR kit, but is not limited thereto as long as it can measure the expression level of miRNA or its target gene.

[0037] In another aspect of the present invention, in order to achieve the above-mentioned object, the present invention provides a method for providing information for the diagnosis of vascular smooth muscle cell proliferative diseases, comprising the steps of: a) measuring the expression level of miRNA in an isolated biological sample; and b) determining that there is a risk of developing a vascular smooth muscle cell proliferative disease if the expression level of the miRNA is increased in a control group.

[0038] The "vascular smooth muscle cell proliferative disorder" and "diagnosis" are as described above. The step of measuring the expression level of miRNA in the isolated biological sample specifically means measuring the expression level of miR-132-3p, miR-370-3p, miR-130b-5p or miR-410-3p, and more specifically means measuring the expression level of miR-370-3p.

[0039] The information providing method may further include a step of determining whether the expression level of a target gene of miRNA in the isolated biological sample is decreased compared to a control group, and specifically, the target gene means BMP7.

[0040] The present invention may also include a preparation for measuring the expression level of the miRNA or its target gene. The preparation for measuring the expression level refers to a preparation that specifically binds to the miRNA or its target gene to enable recognition or amplification. Specific examples include, but are not limited to, an antibody, primer, or probe that specifically binds to the miRNA or its target gene, and a person skilled in the art should be able to select an appropriate preparation to meet the purpose of the invention.

[0041] The preparation may be directly or indirectly labeled for measuring the expression level of the miRNA or its target gene. Specifically, the label may be, but is not limited to, a ligand, a bead, a radioactive nuclide, an enzyme, a substrate, a cofactor, an inhibitor, a fluorescent substance, a chemiluminescent substance, a magnetic particle, a hapten, or a dye. Specific examples of the ligand include biotin, avidin, and streptavidin, the enzyme includes luciferase, peroxidase, and beta-galactosidase, and the fluorescent substance includes, but is not limited to, fluorescein, coumarin, rhodamine, phycoerythrin, and sulforhodamine acid chloride (Texas red). Most of the known labels may be used as such detectable labels, and a person skilled in the art should be able to select an appropriate label to meet the purpose of the invention.

[0042] The term "primer" refers to a short base sequence having a short free 3' hydroxyl group, capable of forming a base pair with a complementary template and serving as a starting point for copying the template strand. In the present invention, the primer used for amplifying the miRNA may be a single-stranded oligonucleotide that can act as a starting point for template-directed DNA synthesis under appropriate conditions (e.g., four other nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature, but the appropriate length of the primer may vary depending on the purpose of use. The primer sequence does not need to be completely complementary to the polynucleotide of the miRNA of the gene or its complementary polynucleotide, and can be used as long as it is sufficiently complementary to hybridize.

[0043] The term "probe" refers to a labeled nucleic acid fragment or peptide capable of specifically binding to miRNA. Specific examples of the probe include oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, oligonucleotide peptide probes, and polypeptide probes.

[0044] In the present invention, the separated biological sample specifically means blood or tubular tissue sections from patients with arteriosclerosis type II and type IV lesions, and the tubular tissue more specifically means tissue scraped from a clogged arterial blood vessel, but is not limited thereto.

[0045] miR-370-3p has been detected in the blood of patients with unstable angina pectoris, type 2 diabetes, and hyperlipidemia, and its high expression was confirmed in tubular tissue sections from patients with atherosclerosis, confirming that it is a new miRNA associated with atherosclerosis.

[0046] The method of the present invention for providing information necessary for determining the presence or absence of onset of a disease may include a step of quantitatively analyzing the expression level of miRNA or its target gene from blood or tubular tissue of an individual suspected of onset of a vascular smooth muscle cell proliferative disease, specifically atherosclerosis.

[0047] The term "individual" can include, without limitation, mammals, including, for example, rats, livestock, humans, etc., that may be affected or have a vascular smooth muscle cell proliferative disorder. In another aspect of the present invention, in order to achieve the above-mentioned object, the present invention can provide a use of a preparation for measuring the expression level of miRNA or a miRNA target gene for producing a diagnostic preparation for a vascular smooth muscle cell proliferative disease.

[0048] The "vascular smooth muscle cell proliferative disorder", "diagnosis" and "miRNA" are as described above. In another aspect of the present invention, in order to achieve the above-mentioned object, the present invention can provide a method for treating a vascular smooth muscle cell proliferative disease, comprising administering to an individual in need thereof a pharmaceutical composition for preventing or treating a vascular smooth muscle cell proliferative disease, the pharmaceutical composition comprising the miRNA expression inhibitor and a pharma- ceutical acceptable carrier.

[0049] The "vascular smooth muscle cell proliferative disease", "prevention", "treatment" and "miRNA" are as described above. In the present invention, the expression inhibitor refers to, but is not limited to, small interfering RNA (siRNA), aptamer, or antisense RNA that binds complementarily to each miRNA.

[0050] In the present application, the term "individual" refers to any animal, including a human, that has or is affected by a vascular smooth muscle cell proliferative disorder of the present invention. The pharmaceutical composition of the present invention can be administered to an individual to provide preventive and therapeutic effects for the disorder.

[0051] The pharmaceutical compositions of the present invention are administered in a pharma- ceutically effective amount. In this application, the term "administration" refers to introducing the pharmaceutical composition of the present invention into a subject by any appropriate method, and the administration route can be through various parenteral routes as long as the target tissue can be reached.

[0052] The pharmaceutical composition can be appropriately administered to an individual according to the purpose or need, according to the usual method, administration route, and dosage used in the art. Examples of administration routes include parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, an appropriate dosage and number of administrations can be selected according to a method known in the art, and the amount and number of administrations of the pharmaceutical composition of the present invention actually administered can be appropriately determined according to various factors such as the type of symptoms to be treated, the administration route, sex, health condition, diet, age and weight of the individual, and the severity of the disease.

[0053] The term "pharmacologically effective amount" in the present invention means an amount sufficient to inhibit or alleviate increased vascular permeability with a reasonable benefit / risk ratio applicable to medical use, and the level of the effective dose can be determined based on factors including the type and severity of the individual, age, sex, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, and other factors well known in the medical field. For example, the dose can be 0.01 to 500 mg / kg, specifically 10 to 100 mg / kg, per day, and the dose can be administered once or in several divided doses per day.

[0054] The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect at the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0055] The composition of the present invention can be used alone for the prevention or treatment of vascular smooth muscle cell proliferative diseases, or can be used in combination with surgery, hormone therapy, drug therapy, and methods using biological response modifiers.

[0056] In another aspect of the present invention, in order to achieve the above-mentioned object, the present invention provides a method for screening target genes of miRNAs that suppress vascular smooth muscle cell proliferation or migration, comprising a step of profiling differentially expressed genes (DEGs) in an injured artery 3 and 5 days after injury.

[0057] The "injured artery," "vascular smooth muscle cells," "proliferation," and "miRNA" are as described above. The screening method specifically includes the steps of: 1) profiling differentially expressed genes (DEGs) in the injured artery at 3 and 5 days after injury; 2) performing in silico target profiling for miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p through a target prediction database using the DEGs; 3) selecting DEGs predicted in common between rats and humans in two or more databases as target genes for each miRNA; and 4) confirming that the selected target genes are down-regulated in the injured artery.

[0058] The profiling in step 1) refers to gene expression profiling, which can simultaneously compare the expression levels of various genes between two or more samples. In analyzing gene expression, northern blotting, microarray analysis, RNA sequencing, etc. can be used, and in the present invention, the overall gene expression change was confirmed through RNA sequencing, but the present invention is not limited thereto.

[0059] The target prediction database in step 2) is for predicting the target genes of miRNAs, and the function of miRNAs is generally known through the genes regulated by miRNAs. As of October 2018, 38,589 human miRNAs (mature miRNAs) have been databased (miRBase database, version 22.1). The 3'UTR of the target gene and the seed of the miRNA can be aligned to predict, and the prediction accuracy is improved by adding structural features or evolutionarily conserved binding sites. Programs that provide such prediction results include DIANA-microT, MicroInspector, MiRanda, PicTar, RNA22, RNAhybrid, TargetBoost, TargetScan, miRDB, miRmap, etc. In the present invention, TargetScan, miRDB, and miRmap are used, but the present invention is not limited thereto.

[0060] The target gene in step 3) specifically means a gene whose intracellular expression can be regulated through complementary binding between miRNA and mRNA, and one miRNA has many target genes.

[0061] The screening in step 4) refers to genetic screening, which means selecting a target gene present in a library. Screening methods include screening using hybridization, screening using antibodies, screening using differences in gene expression, screening using binding with specific proteins, etc., and in the present invention, screening using differences in gene expression is used, but is not limited thereto. EXAMPLES

[0062] The present invention will be described in more detail with reference to the following examples. These examples are provided to more specifically explain the present invention, and are not intended to limit the scope of the present invention.

[0063] Experimental Example 1. Preparation of a carotid artery balloon injury model After basic breeding, 10-week-old male albino rats (Sprague-Dawley rats) were adapted to the environment for one week, and then a carotid artery balloon injury model was prepared.

[0064] Specifically, balloon injury was created in the left common carotid artery of rats using a Fogarty balloon embolectomy catheter. The left external carotid artery was exposed and the peripheral arterial branches were electrocoagulated, after which a catheter was inserted through a transverse incision in the external carotid artery, positioned 1 cm medial to the cut site, inflated, and moved back and forth along the common carotid artery to injure the carotid artery. For catheter-mediated intra-arterial delivery of miRNA inhibitors, the transfection complex (200 nM miRNA / 10 μl Lipofectamin RNAimax in 200 μl Opti-MEM) was injected into the lumen of the injured carotid artery and incubated for 15 min. Before removing the catheter, the lumen was washed once with saline, and after removing the catheter, the punctured site was sealed and the clap of the common carotid artery was released to re-establish blood flow. Unless otherwise stated, rats were then allowed to recover in their cages for up to 14 days.

[0065] Experimental Example 2. Histopathological analysis Balloon-injured rats were anesthetized and transcardiac perfusion-fixation was performed with 3.7% formaldehyde in heparinized saline, after which the common carotid artery was excised. The vessels were embedded in paraffin and cut with a rotary microtome (Leica RM2255). Two serial tissue sections (4 μm thick) were obtained from the midsection of the common carotid artery and stained with haematoxylin & eosin. The lumen, internal elastic lamina, and external elastic lamina areas were measured using NIH Image v 1.62. The intimal and medial areas were determined by subtracting the lumen area from the internal elastic area and the internal elastic area from the external elastic area, respectively. For analysis, the values ​​obtained from two serial sections per rat were averaged.

[0066] Experimental example 3. RNA sequencing For small RNA and mRNA sequencing, total RNA was isolated from rat carotid artery tissues using QIAzol Lysis Reagent (Qiagen) according to the manufacturer's protocol. For the sham control group, three carotid artery tissues were pooled to increase the amount of total RNA. The purity and integrity of the total RNA extracts were measured using a NanoDrop 8000 spectrophotometer (Thermo Scientific) and a Bioanalyzer (Agilent Technologies), respectively, and samples with RIN values ​​greater than 8 were used in the sequencing process.

[0067] First, small RNA libraries were prepared for small RNA sequencing using the TruSeq Small RNA Prep kit (Illumina). Briefly, 1 μg of total RNA was ligated with 3' and 5' RNA adaptors. Reverse transcription PCR was performed using primers annealing to the 3' and 5' adaptors to generate and enrich cDNA constructs. The cDNA libraries were purified using the Pippin prep electrophoresis platform (Sage Science), and the quality of the libraries was verified using a 2100 Bioanalyzer (Agilent Technologies). Small RNA sequencing was performed using the Hiseq 2500 system (Illumina) with settings of 1X51.

[0068] Next, mRNA sequencing libraries were prepared for mRNA sequencing using the TruSeq RNA Prep kit v2 (Illumina). Briefly, mRNA samples were purified from 1 μg of total RNA using poly-T oligo-attached magnetic beads, and fragmented mRNA was primed with random hexamers and reverse transcribed. The mRNA template was removed, and second-strand cDNA was synthesized. After 3' A-tailing and 5' end repair, DNA sequencing adapters were ligated to the cDNA template. Then, PCR was performed to amplify the template. The quality of the libraries was verified with a 2100 Bioanalyzer (Agilent Technologies), and mRNA sequencing was performed using a Hiseq 2500 system (Illumina) at 2X101 settings.

[0069] Experimental example 4. Cell culture Primary human aortic SMCs (HASMCs) were purchased from Lonza and were subcultured and expanded in smooth muscle cell growth medium (SmGM) containing 5% fetal bovine serum with growth factors and antibiotics (cat. No. cc-4149, Lonza). Human vascular smooth muscle cells were mainly used for experiments at passage numbers 5 to 7. HEK293T cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin, and all cultures were maintained in a humidified incubator containing 5% CO2 at 37°C.

[0070] mirVana miRNA mimics or inhibitors (Invitrogen, USA) and siRNAs (Bioneer, Korea) were transfected using Lipofectamine RNAimax (Invitrogen).

[0071] Cells were plated in a 6-well plate at 1 x 10 5After 24 h, transfection complexes containing miRNA mimics (0.1 nM each), miRNA inhibitors (10–150 nM each) or siRNAs (100 nM each) were added to the culture plates, and then the culture medium was replaced with fresh medium 24 h after transfection.

[0072] Experimental Example 5: Real-time quantitative PCR Total RNA was isolated from cultured human vascular smooth muscle cells and rat carotid artery tissues using QIAzol Lysis Reagent (Qiagen) according to the manufacturer's protocol. For miRNA validation, individual cDNAs were synthesized from 40 ng of isolated total RNA using the TaqMan MicroRNA Reverse Transcription Kit and specific RT primers from TaqMan MicroRNA Assays (Applied Biosystems). Mature miRNA expression levels were quantified through quantitative real-time PCR using TaqMan Universal Master Mix II and specific FAM-based probes and primers from TaqMan MicroRNA Assays (Applied Biosystems) according to the manufacturer's protocol.

[0073] The thermal cycling conditions for miRNA were as follows: initial enzyme activation at 95°C for 10 minutes, followed by denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, and extension, repeated 40 times for amplification.

[0074] U87, snoRNA and U6 were used as internal controls for rat carotid arteries, and U6 was used as an internal control for human vascular smooth muscle cells. For mRNA validation, reverse transcription was performed on 1 μg of isolated total RNA using the ImProm-II RT system (Promega). Quantitative real-time PCR was performed using gene-specific primers (all Qiagen) and SYBR Green (Roche).

[0075] The thermal cycling conditions for mRNA were as follows: initial denaturation at 95° C. for 15 min, followed by 40 cycles of denaturation at 94° C. for 15 s, annealing at 55° C. for 30 s, and extension at 72° C. for 30 s for amplification.

[0076] Melting curve analysis was performed at the end of PCR, using β-actin or 18S RNA as housekeeping genes. Mature miRNA and transcript levels were detected using the CFX Connect real-time PCR detection system (Bio-Rad). Relative gene expression was determined by ΔΔCt values.

[0077] Experimental Example 6: Immunoblot analysis Human vascular smooth muscle cells were washed twice with cold phosphate-buffered saline (PBS) and quickly frozen in liquid nitrogen. The cells were lysed in a lysis buffer containing 20 mM HEPES (pH 7.0), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM EDTA (pH 8.0), 2 mM EGTA (pH 8.0), 1 mM DTT, 5 mM Na3VO4, 5 mM NaF, 1 mM AEBSF, 5 μg / ml aprotinin, and 5 μg / ml leupeptin. After centrifugation at 12,000×g for 10 min, the purified lysates were separated on a denaturing polyacrylamide gel (30 μg each) and transferred to a nitrocellulose membrane. The membrane was incubated overnight with primary antibodies in Tris-buffered saline (TBS) solution containing 0.05% Tween®-20 and 5% BSA at 4°C. The membrane was then incubated for 1 h with HRP-conjugated secondary antibody diluted 1:3000 in TBS solution containing 0.05% Tween®-20 and 5% skim milk. Immunoreactive bands were visualized using WESTSAVE up ECL solution (cat. No. LF-QC0101, Abfrontier, Korea).

[0078] Experimental Example 7: Cell proliferation and cell cycle analysis For cell proliferation analysis, miRNA inhibitor-transfected human vascular smooth muscle cells (HASMCs) were seeded at a density of 2,000 cells / well in 96-well plates and grown for the indicated times, followed by incubation with WST-1 reagent (10 μl / well) for 1 h at 37° C. The number of viable cells was estimated by measuring the absorbance at 450 nm.

[0079] For cell cycle analysis, human vascular smooth muscle cells (1 × 10 5 Cells) were harvested 48 h post-transfection. The cells were fixed, permeabilized with 70% ethanol overnight at -20°C, treated with 100 μg / ml RNase A for 1 h at 37°C, and stained with 10 μg / ml propidium iodide. Intracellular DNA content was measured with a FACSCalibur system (BD biosciences), and the percentage of G0 / G1 diploid cells was analyzed using Modfit LT software (Verity Software House).

[0080] Experimental example 8: Immunofluorescence staining For histological staining, paraffin sections of balloon-injured carotid arteries were deparaffinized with xylene and rehydrated with ethanol. The rehydrated tissue sections were boiled for 20 min in citrate-based antigen unmasking solution (Vector Laboratories) for antigen retrieval. For double immunofluorescence, the tissue sections were blocked for 1 h with 5% normal donkey serum in PBS-T (0.3% Triton X-100 in PBS). The samples were then incubated with FITC-conjugated anti-vWF antibody (1:50 dilution, Abcam) overnight at 4°C. After washing three times with PBS-T, the samples were incubated with Cy3-conjugated anti-SMA antibody (1:200 dilution, Sigma Aldrich) at room temperature in the dark for 2 h.

[0081] For F-actin staining, human vascular smooth muscle cells were fixed with 3.7% formaldehyde for 15 min and permeabilized with PBS-T for 15 min at room temperature. The cells were labeled with Alexa Fluor 488-conjugated phalloidin (cat. no A12379, Invitrogen) for 60 min at room temperature.

[0082] For TUNEL analysis, the fixed cells were incubated with permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) at 4°C for 2 minutes and then incubated with the TUNEL reaction mixture from the In Situ Cell Death Detection Kit (Roche Diagnostics) at 37°C for 60 minutes.

[0083] Nuclear DNA was labeled with DAPI, and fluorescent images were obtained using an LSM880 Airyscan confocal microscope (Carl Zeiss). Experimental example 9: Transwell migration assay Chemotactic cell migration was measured using 24-well Transwell culture chambers (Costar; 8 mm pore size). The upper chamber was coated with gelatin B (1 mg / ml) and air-dried for 1 h. Human vascular smooth muscle cells were transfected with miRNA inhibitors for 24 h, serum-starved for 18 h, and then replated in the upper chamber at a density of 6,000 cells / chamber in basal medium. Complete medium was added to the lower chamber, and the Transwell chamber was incubated at 37°C for 24 h. Non-migrating cells from the upper side of the membrane were removed, and cells that had passed through and attached to the lower side of the membrane were fixed and stained with 0.6% hematoxylin and 0.5% eosin. The number of stained cells in four sections was counted and averaged.

[0084] Experimental Example 10: Monocyte adhesion assay Human vascular smooth muscle cells were transfected with miRNA inhibitors for 24 hours and then plated again in a 96-well plate (4,000 cells / well). The human vascular smooth muscle cells were then stimulated with TNF-α (10 ng / ml) for 18 hours. Separately, monocytic U937 cells (1 × 10 6 The activated U937 cells (1 × 10 cells / well) were stimulated with IFN-γ (50 μg / ml) for 24 hours. The activated U937 cells were labeled with 4 μM tetramethylrhodamine ethyl ester, perchlorate (cat. No. T-669, Molecular Probes) for 30 minutes, and the labeled U937 cells (1 × 10 5 The mixture (100 x 100 cells / well) was added to confluent human vascular smooth muscle cells and incubated for 1 h at 37°C. Unbound U937 cells were gently removed by washing three times with PBS, and attached U937 cells were detected and counted using a ZOE Fluorescent Cell Imager (Bio-Rad).

[0085] Experimental Example 11: Luciferase reporter assay For prediction of target genes of miR-370-3p, reporter plasmid pMirTarget containing the full-length 3'UTR sequence of human BMP7 gene (hBMP7-3'UTR) was purchased from Origene (cat. No. SC218118). Two consecutive nucleotides in the miR-370-3p target region #1 (nucleotides 229-235) of hBMP7-3'UTR were mutated as a negative control. For reporter analysis, HEK293T cells were plated in 24-well plates and transfected with reporter plasmid for 6 hours. Then, the cells were transfected with miR-370-3p mimic for 48 hours, lysed and analyzed for protein. Identical amounts of cell lysates were applied for luciferase analysis. Luminescence signals were measured with a VICTOR Multilabel Plate Reader (Perkin Elmer).

[0086] Experimental Example 12: In situ hybridization miR-370-3p expression in arterial tissue was detected using miRCURY LNA miRNA in situ hybridization (ISH) Optimization Kits (Qiagen) according to the manufacturer's protocol. 5' and 3' DIG-modified has-miR-370-3p miRCURY LNA miRNA Detection Probes were used for hybridization. Briefly, 4 μm paraffin sections of fixed arterial tissue were deparaffinized and rehydrated. Nucleases were inactivated with proteinase K for 10 min at 37°C, and hybridization was performed with miRNA detection probes (40 nM each) by incubation at 30°C below the RNA Tm of the probes for 1 h. Slides were washed with serial dilutions of saline-sodium citrate (SSC) hybridization buffer at each annealing temperature, and immunodetection was performed by incubating alkaline phosphatase-conjugated anti-DIG antibody (cat. No. 11093274910, Roche) for 60 min at room temperature. The slides were incubated with NBT / BCIP (cat. No. 11697471001, Roche) substrate solution containing 0.2 nM Levamisol for 2 h at 37°C for color development. Serial tissue sections annealed with scramble probes were used as controls, and cell nuclei were labeled with Nuclear Fast Red.

[0087] Experimental Example 13: Statistical Analysis Unless otherwise stated, data were analyzed by one-way ANOVA with Student's t-test between two groups and Turkey's test for different groups. P<0.05 was considered statistically significant.

[0088] Example 1: Identification of differentially expressed miRNAs in injured arteries Based on the histological analysis of neointimal thickening according to injury time in a rat carotid artery balloon injury model investigated in a previous study, we selected two time points, 3 and 5 days after balloon injury, to identify miRNAs that direct the initiation of smooth muscle cell reverse differentiation and proliferation.

[0089] Specifically, total RNA samples were prepared and separated into small RNAs and messenger RNAs through appropriate purification procedures. Both RNA pools were subjected to library construction and HiSeq-based sequencing, and the small RNA sequencing data was first analyzed using RSEM software.

[0090] As a result, as shown in Figure 1, 62 miRNAs were differentially expressed at the two time points compared to the sham control group (Figure 1A). The miRNAs were examined one by one for their novelty as well as their relevance to the vasculature, and 50 miRNAs were selected for quantitative validation by miRNA-specific real-time PCR. As a result, 12 upregulated miRNAs and 6 downregulated miRNAs showed more than 5-fold changes in the injured artery compared to the sham control group, and 1 / 5 of the miRNAs were temporarily downregulated 3 days after injury, implying their potential as early-response miRNAs. Some miRNAs that were not confirmed in the human database or had no seed sequence identity between rats and humans were excluded. Among the remaining miRNA candidates, miRNAs such as miR-221-3p, miR-21-5p and miR-146a-5p were known to mediate smooth muscle cell hyperplasia {Sun, 2011 #2627;Liu, 2009 #2623;Ji, 2007 #2625}, supporting the validity of the selection strategy using rodent models.

[0091] The top 10 miRNAs, including 8 upregulated and 2 downregulated miRNAs, showed dramatic expression changes of more than 5-fold in injured carotid arteries (Figure 1B). To examine their human relevance, we confirmed their expression levels in cultured primary human vascular smooth muscle cells with a synthetic phenotype, and confirmed through qPCR analysis that 6 of the 8 upregulated miRNAs were significantly expressed in human smooth muscle cells (Figure 1C).

[0092] Thus, through miRNA screening using rodent models, we discovered a subset of miRNAs, including some known candidates associated with neointimal proliferation. Example 2. Upregulated miRNAs that regulate smooth muscle cell function in vitro To determine the role of upregulated miRNAs in smooth muscle cell biology, three types of cell-based assays were performed in vitro using human vascular smooth muscle cells transfected with miRNA inhibitors.

[0093] In smooth muscle cell proliferation, miR-132-3p and miR-370-3p showed significant inhibitory effects (Figure 2A), and monocyte adhesion to human vascular smooth muscle cells, a sign of inflammation, was significantly inhibited by three miRNAs, miR-130b-5p, miR-132-3p, and miR-410-3p. Based on these cell analyses, we performed a deeper investigation of proliferation-related miRNAs, namely, miR-132-3p and miR-370-3p. Both miRNA inhibitors induced cell cycle arrest at the G1 phase and persistently inhibited the time-dependent proliferation of human vascular smooth muscle cells (Figures 2B and 2C), and consistent with this, the levels of cyclin-dependent kinase inhibitors p21 and p27 were significantly increased by both miRNA inhibitors (Figure 2D). However, such miRNA inhibition was not accompanied by cell apoptosis in human vascular smooth muscle cells, suggesting cell proliferation inhibition of smooth muscle cell proliferation.

[0094] In injured arterial tissue, the phenotypic transition of smooth muscle cells from a contractile to a synthetic state through retrodifferentiation must precede proliferation-dependent neointimal hyperplasia, and we therefore investigated whether miR-132-3p and miR-370-3p are involved in the phenotypic transition.

[0095] As the simplest method to mimic the in vivo arterial environment, human vascular smooth muscle cells were induced to have a contractile phenotype by culturing them at high confluence on laminin-coated plates. When high confluence smooth muscle cells were re-plated at low confluence, the cells were gradually transformed into a synthetic phenotype. Through immunoblot analysis in Experiment 6, it was confirmed that transfection of miR132-3p inhibitors restored the SMA levels that disappeared due to low confluence (Figure 2E). Indeed, immunofluorescence staining of F-actin filaments demonstrated that the synthetic phenotype of human vascular smooth muscle cells at low confluence was transformed into a contractile phenotype by miR-132-3p inhibitors (Figure 2F).

[0096] Thus, we confirmed that miR-132-3p and miR-370-3p may regulate phenotypic transition and proliferation signals, respectively. Example 3. Upregulated miRNAs regulating neointimal proliferation in vivo To evaluate the efficacy of miRNAs in vivo using a balloon injury model, the arterial expression levels of four functional miRNAs were examined by in situ hybridization as in Experimental Example 12 above.

[0097] As a result, the staining images showed that the expression of two proliferation-related miRNAs (miR-132-3p and miR-370-3p) was significantly increased in the balloon-injured carotid artery compared to the sham control group (Figure 3A), and the other two inflammation-related miRNAs (miR-130b-5p and miR-410-3p) were also substantially induced by balloon injury. The in vivo functions of the four miRNAs were evaluated in balloon-injured rat arteries.

[0098] Catheter-mediated local intramural delivery of miRNA inhibitors significantly reduced neointimal proliferation in balloon-injured lesions compared with the control group (Figure 3B), and immunofluorescence staining of carotid arteries for von Willebrand factor (vWF) and SMA as EC and smooth muscle cell markers showed that miR-130b-5p inhibitors promoted the recovery of the EC monolayer, so-called re-endothelialization (Figure 3C), suggesting that miR-130b-5p exerted an antiproliferative function in ECs but not in smooth muscle cells.

[0099] Through this, it was confirmed that the four miRNAs play a significant role in arterial homeostasis, such as vascular inflammation and proliferation. Example 4. Confirmation of target genes of functional miRNAs Next, to understand the biological consequences of the miRNAs selected in Example 3, we performed mRNA sequencing to profile the differentially expressed genes (DEGs) in the balloon-injured rat carotid arteries.

[0100] As a result, as shown in Figure 4, 3,699 DEGs were confirmed to have been significantly changed 3 and 5 days after injury compared to the sham control group (Figure 4a). Heat map analysis showed that the upregulated and downregulated DEGs were half and half, respectively (Figure 4b). Using these DEGs, in silico target profiling was performed for the selected miR-132-3p / miR-370-3p and miR-130b-5p / miR-410-3p through target prediction databases (TargetScan, miRDB, and miRmap), and DEGs that showed high prediction scores (TargetScan<-0.1, miRDB>70, miRmap>70) in common for both rats and humans in one or more databases were selected as predicted targets of each miRNA. Gene Ontology (GO) enrichment analysis further identified target gene pools functionally associated with cognate miRNAs, and then real-time PCR confirmed the down-regulated genes in balloon-injured carotid arteries compared to sham controls (Figure 4c). For human relevance, we further investigated the expression of the above-mentioned representative genes in human vascular smooth muscle cells transfected with miRNA inhibitors.

[0101] As a result, the core target genes for each miRNA were identified through real-time PCR, and it was confirmed that SOCS2, BMP7, TSPAN2, and SMAD6 were specific targets of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p, respectively (Figure 4d).

[0102] Example 5. MiR-370 / BMP7 axis essential for smooth muscle cell proliferation In particular, because miR-370 has been detected in the blood of patients with unstable angina, type 2 diabetes, and hyperlipidemia [Hoekstra, 2010 #2635; Motawae, 2015 #2636; Gao, 2012 #2637], we attempted to detect miR-370 in human cells and arterial tissues to investigate the biological significance of the miR-370 / BMP (bone morphogenic protein)-7 axis in smooth muscle cell proliferation.

[0103] High expression of miR-370-3p was confirmed in tubular tissue sections from human patients with arteriosclerosis type II and type IV lesions through in situ hybridization (Figure 5a), confirming that miR-370 is a new miRNA associated with atherosclerosis. We then investigated the relationship between miR-370 and BMP7 in cultured human vascular smooth muscle cells, and indeed, serum stimulation induced the expression of miR-370-3p, but conversely reduced the expression of BMP7 (Figure 5b). Consistent with this, reporter analysis using the 3-UTR region of the human BMP7 gene demonstrated that the expression of luciferase containing the wild-type UTR, but not the mutated UTR, was indeed reduced by miR-370 mimics (Figure 5d). Western blot analysis also clearly demonstrated that miR-370 inhibition increased the level of BMP7 protein in human smooth muscle cells (Figure 5d).

[0104] These results collectively confirmed that the BMP7 gene is a bona fide miR-370 target in human smooth muscle cells. Because recombinant BMP7 treatment was previously reported to suppress smooth muscle cell proliferation [Lagna, 2007 #2641; Dorai, 2000 #2640], we investigated the involvement of the miR-370 / BMP7 axis in smooth muscle cell proliferation. As direct evidence, BMP7 treatment markedly induced SMAD1 / 5 / 9 phosphorylation in human vascular smooth muscle cells (Figure 5E), suggesting an antimitotic effect of BMP7 in smooth muscle cells. Moreover, cell proliferation analysis demonstrated that BMP7 depletion restored smooth muscle cell proliferation suppressed by miR-370 inhibitors (Figure 5F), thus confirming that miR-370-dependent regulation of BMP7 expression is a prerequisite for smooth muscle cell growth and neointimal proliferation occurring in injured arteries.

[0105] In summary, the miRNA inhibitors of the present invention, specifically, the inhibitors of miR-132-3p, miR-370-3p, miR-130b-5p and miR-410-3p, have a proliferation-inhibitory effect on smooth muscle cells, and thus it can be predicted that the incorporation of the miRNA inhibitors in smooth muscle cells will be effective in preventing or treating vascular smooth muscle cell proliferative diseases.

[0106] From the above description, it will be understood by those skilled in the art to which the present invention pertains that the present invention may be embodied in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A pharmaceutical composition for preventing or treating a vascular smooth muscle cell proliferative disease, comprising an miRNA (microRNA) inhibitor as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the miRNA is one or more selected from the group consisting of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p.

3. 2. The pharmaceutical composition of claim 1, wherein the miRNA inhibitor regulates vascular smooth muscle cell function in vitro and neointimal proliferation in vivo by suppressing miRNAs up-expressed in injured arteries.

4. The pharmaceutical composition according to claim 1, wherein the miRNA inhibitor is at least one selected from the group consisting of siRNA, aptamer, antisense oligonucleotide, ribozyme and compound specific to miRNA.

5. 2. The pharmaceutical composition according to claim 1, wherein the vascular smooth muscle cell proliferative disease is a disease selected from the group consisting of vascular stenosis, vascular restenosis, atherosclerosis, heart failure, myocardial infarction, angina pectoris, arrhythmia, hypertensive heart disease, congenital heart disease, stroke and peripheral vascular stenosis.

6. A diagnostic kit for vascular smooth muscle cell proliferative disorders, comprising a preparation capable of detecting miRNA.

7. The kit of claim 6, wherein the miRNA is selected from the group consisting of miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p.

8. The kit according to claim 6, wherein the kit is selected from the group consisting of a microarray, an aptamer chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a serial analysis of gene expression (SAGE) kit, a quantitative real time PCR (qRT-PCR) kit, or a combination thereof.

9. Measuring the expression level of miRNA in the isolated biological sample; and The method for providing information on diagnosis of vascular smooth muscle cell proliferative disorders includes a step of determining that a subject is at risk of developing a vascular smooth muscle cell proliferative disorder when the expression level of the miRNA is increased in a control group.

10. The method for providing information according to claim 9, wherein the miRNA is selected from the group consisting of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p.

11. The method of claim 9, further comprising determining whether the expression level of a miRNA target gene in the biological sample is decreased compared to a control group.

12. 9. The method for providing information according to claim 8, wherein the separated biological sample is blood or a tubular tissue section from an arteriosclerosis patient.

13. A method for treating a vascular smooth muscle cell proliferative disease, comprising administering to an individual, other than a human, in need thereof a pharmaceutical composition for preventing or treating a vascular smooth muscle cell proliferative disease, the pharmaceutical composition comprising an miRNA expression inhibitor and a pharma- ceutical acceptable carrier.

14. The method of claim 13, wherein the miRNA is selected from the group consisting of miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p.

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