Compositions for the diagnosis, prevention, or treatment of vascular smooth muscle cell proliferation 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 containing an miRNA (microRNA) inhibitor as an active ingredient; a kit for diagnosing a vascular smooth muscle cell proliferative disease containing a preparation capable of detecting miRNA; a method for providing information for diagnosing the disease; and a treatment method.

Background Art

[0002] Normal arterial blood vessels are composed of two main cell types: a monolayer of healthy endothelial cells (ECs) and contractile smooth muscle cells (smooth muscle cells). However, the internal accumulation of low-density lipoprotein combined with blood flow disorders can cause inflammation in ECs. Monocytes / macrophages adhere to inflamed EC lesions, penetrate the EC monolayer, and absorb lipid particles to become foam cells. The generated macrophage-driven foam cells and immune cells in atherosclerotic lesions secrete many growth factors and cytokines that stimulate smooth muscle cells for dedifferentiation and fibrous cap formation. The entire atherosclerotic process, including the initial inflammatory reaction, ultimately leads to the occlusion of the arterial lumen. Currently, the only way to restore occluded arteries in patients with arteriosclerosis is to perform angioplasty and stent surgery in parallel. However, after the surgery, thrombosis, which is the main complication due to endothelial damage, occurs, and this induces the proliferation of additional neointima called in-stent restenosis. This process of restenosis complications is similar to the proliferation and migration of smooth muscle cells that have been dedifferentiated in the late stage of arteriosclerosis again. Therefore, this suggests that no target approach can be the correct solution for preventing arteriosclerosis.

[0003] MicroRNAs (abbreviated as miRNAs) were discovered in 1993 while studying the lin-4 gene in nematodes, and their function as translation regulators has attracted much attention over the past few decades. A unique characteristic of miRNAs is their ability to simultaneously regulate the expression of various genes in the 3'UTR, and indeed, various studies have attempted to identify 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. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Against this background, the inventors diligently conducted research to screen for vascular miRNAs associated with the overproliferation and migration of arterial smooth muscle cells. As a result, using a standard animal model exhibiting smooth muscle cell proliferation, they 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 novel miRNA associated with a very important proliferative disorder of smooth muscle cells 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 the prevention or treatment of vascular smooth muscle cell proliferative disorders, comprising a 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 that includes a formulation 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 vascular smooth muscle cell proliferative disorders, comprising administering the pharmaceutical composition containing a miRNA expression inhibitor to individuals other than humans who require it. [Effects of the Invention]

[0007] This invention identifies subsets of miRNAs involved in proliferative disorders of vascular smooth muscle cells, namely miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p. These miRNAs show a significant increase in expression of more than five times in damaged arteries compared to the control group. In particular, high expression of miR-370-3p has been confirmed in the tubular tissue of patients with arteriosclerosis, making it useful for diagnosing the aforementioned disease. Furthermore, it can be utilized for the prevention or treatment of proliferative disorders of vascular smooth muscle cells. [Brief explanation of the drawing]

[0008] [Figure 1a] This is a heatmap analysis of miRNAs that showed differential expression at 3 and 5 days after arterial injury compared to a sham control group in an animal model of balloon-induced arterial injury. [Figure 1b] This graph shows eight upregulated miRNAs and two downregulated miRNAs that showed a five-fold or greater change in expression levels among the differentially expressed miRNAs. [Figure 1c] This graph confirms that 6 out of the 8 upregulated miRNAs in Figure 1b were significantly expressed in human smooth muscle cells. [Figure 2a] To confirm the role of upregulated miRNAs, we used human vascular smooth muscle cells transfected with miRNA inhibitors. The results showed that miR-132-3p and miR-370-3p had a significant inhibitory effect on smooth muscle cell proliferation, and miR-130b-5p, miR-132-3p, and miR-410-3p significantly inhibited monocyte adhesion. [Figure 2b]This graph shows that miR-132-3p and miR-370-3p inhibitors induce cell cycle arrest at the G1 stage of the cell cycle, thereby suppressing time-dependent proliferation of human vascular smooth muscle cells. [Figure 2c] This graph shows that miR-132-3p and miR-370-3p inhibitors induce cell cycle arrest at the G1 stage of the cell cycle, thereby suppressing time-dependent proliferation of human vascular smooth muscle cells. [Figure 2d] This graph shows that miR-132-3p and miR-370-3p inhibitors significantly increase the levels of cyclin-dependent kinase inhibitors p21 and p27. [Figure 2e] This graph confirms that miR-132-3p is involved in smooth muscle cell phenotypic transition, as miR-132-3p inhibitors restore SMA levels, leading to a conversion of smooth muscle cells to a contractile phenotype. [Figure 2f] This graph reconfirms that the synthetic phenotype of human vascular smooth muscle cells was altered to a contractile phenotype by a miR-132-3p inhibitor via F-actin filaments. [Figure 3a] In situ hybridization of four selected miRNAs revealed that their arterial expression levels were significantly increased in balloon-damaged arteries, while miR-130b-5p and miR-410-3p were induced by balloon damage. [Figure 3b] These photographs and graphs confirm that catheter-mediated local intramural transmission of four selected miRNA inhibitors significantly reduced neointimal proliferation in balloon-injured lesions compared to the control group. [Figure 3c] Among miRNAs, the miR-130b-5p inhibitor exhibits antiproliferative efficacy in the EC by promoting the recovery of the EC monolayer through re-endothelialization. [Figure 4a]This profile identifies differentially expressed genes (DEGs) in damaged arteries. [Figure 4b] The upward and downward controlled DEG were confirmed through heatmap analysis. [Figure 4c] This graph shows that, via real-time PCR, the predicted target genes of the four selected miRNAs were downregulated in balloon-injured carotid arteries compared to the sham control group. [Figure 4d] This graph confirms 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, through real-time PCR. [Figure 5a] High expression of miR-370-3p was confirmed in tubular tissue sections from human patients with type II and type IV arteriosclerosis lesions. [Figure 5b] This study investigated the relationship between miR-370-3p and BMP7 through serum stimulation. Serum stimulation induced miR-370-3p expression but decreased BMP7 expression. [Figure 5c] We confirmed that BMP7 protein levels increase in vascular smooth muscle cells through miR-370-3p inhibition. [Figure 5d] This study confirmed that the miR-370-3p mimetic reduces luciferase expression in a non-negative control group containing wild-type UTRs in which two consecutive nucleotides within the miR-370-3p target region of human BMP7-3'UTR have been mutated. [Figure 5e] This study confirmed the induction of SMAD1 / 5 / 9 phosphorylation in vascular smooth muscle cells by BMP7 treatment. [Figure 5f] This study demonstrated that siRNA transfection reduces BMP7 levels, thereby restoring smooth muscle cell proliferation that had been suppressed by miR-370-3p inhibitors.

Best Mode for Carrying Out the Invention

[0009] Specifically, it is as follows. However, it is not considered that the scope of the present invention is limited by the following specific descriptions. That is, each of the explanations and embodiments disclosed in the present invention can be applied to different explanations and embodiments, and all combinations of various elements disclosed in the present invention belong to the scope of the present invention.

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

[0011] In addition, the present invention provides use of a miRNA (microRNA) inhibitor for preventing or treating a vascular smooth muscle cell proliferative disease. In the present application, the term "miRNA" can be described as microRNA, microRNA, etc., and is a small RNA that plays a role in controlling gene expression in organisms. Specifically, it is a small RNA containing 20 to 25 nucleotides that plays an important regulatory role in the gene expression process by suppressing the translation of target mRNA through complementary base pair sequences with the 3'UTR (untranslated region) of the target mRNA. The miRNA plays an important role in cell functions including proliferation, differentiation, apoptosis, etc., and is an evolutionarily conserved regulatory substance present in all animals. Some miRNAs can regulate gene expression through epigenetic regulatory mechanisms (such as histone modification, DNA methylation, etc.) associated with their promoter sites.

[0012] The miRNAs used in this invention regulate smooth muscle cell overgrowth, phenotypic translocation, and migration. Specifically, miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p were identified through a balloon-damaged artery model. Furthermore, these core target genes were further identified, and in particular, miR-370-3p was discovered to be a novel miRNA associated with atherosclerosis, a finding first elucidated by the inventors.

[0013] In this invention, the term "miRNA inhibitor" means a preparation that reduces the expression or activity of each miRNA within a cell, and means blocking miRNAs that either act directly on the said miRNA or indirectly on higher regulatory factors to regulate the expression of target genes. In this invention, the miRNA inhibitor means regulating vascular smooth muscle cell function and in vivo neointimal proliferation by suppressing miRNAs upexpressed in damaged arteries, and specifically means, but is not limited to, inhibitors of miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p, respectively.

[0014] In the present invention, the miRNA suppression can be performed using inhibitors specific to each of miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p. Specifically, these may be, but are not limited to, siRNA aptamers, antisense oligonucleotides, ribozymes, or compounds specific to each miRNA. For the purposes of the present invention, any compound that suppresses miRNAs associated with in vivo neoplastic intima-proliferation can be used.

[0015] The aforementioned damaged arteries were artificially damaged in order to screen for vascular miRNAs whose expression is associated with the overproliferation of arterial smooth muscle cells, for the purposes of the present invention. Specifically, a model was created in which rat carotid arteries were damaged via balloons and used in the experiment.

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

[0017] The aforementioned term, "upregulated," means an increase in mature miRNAs due to increased transcription and processing of specific miRNAs. In this invention, this means increased biosynthesis in the injured artery model compared to the control group. For the purposes of this invention, increased expression specifically means a significant increase of more than five times compared to the control group, but is not limited to this.

[0018] In this application, the term "vascular smooth muscle cells" refers to cells that make up the inner wall of blood vessels and function to maintain constant blood pressure through contraction and relaxation. In normal blood vessels, smooth muscle cells differentiate and produce proteins necessary for contraction and relaxation, stopping cell proliferation. However, if functional problems occur and they dedifferentiate, the blood vessels narrow, leading to diseases such as arteriosclerosis and 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 the increase in the number of organisms or tissue cells through cell division, and usually means an increase in the number of cells within the body of a multicellular organism. 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 arteriosclerotic lesions.

[0020] In this application, the term "vascular smooth muscle cell proliferative disorder" means a disease caused by excessive proliferation of vascular smooth muscle cells. The vascular smooth muscle cell proliferative disorder may include not only vascular stenosis, restenosis, atherosclerosis, and arteriosclerosis that directly result from the proliferation of vascular smooth muscle cells, but also cardiovascular diseases that are secondarily induced or worsened by these diseases, such as heart failure, myocardial infarction, angina pectoris, arrhythmia, hypertensive heart disease, congenital heart disease, stroke, or peripheral vascular stenosis.

[0021] In the present invention, the vascular smooth muscle cell proliferative disease may specifically be vascular stenosis, vascular restenosis, atherosclerosis, or arteriosclerosis. Vascular stenosis is a disease in which the inside of a blood vessel becomes abnormally narrowed due to inflammation, hemoptysis, excessive proliferation of smooth muscle cells, etc., after damage to the blood vessel wall, resulting in a decrease in blood flow. Vascular restenosis is the recurrence of vascular stenosis and often occurs after vascular surgery to resolve vascular stenosis, such as dilating the lumen of a blood vessel or reopening a blocked vessel. Vascular surgeries such as angioplasty (stenting / balloon angioplasty) and coronary artery bypass surgery (vascular bypass or vascular graft) can damage blood vessels or cause inflammation and vascular stenosis during the surgery itself. Atherosclerosis is a disease in which fat is deposited or fibrosis occurs in the inner lining of arteries, and it is known that vascular restenosis that occurs after stent insertion is entirely due to the proliferation, migration, and extracellular matrix secretion of vascular smooth muscle cells, due to the progression of atherosclerosis and vasodilation.

[0022] In the present invention, "prevention" means any action that suppresses or delays the onset of vascular smooth muscle cell proliferative disease by administering the pharmaceutical composition according to the present invention, and "treatment" means any action that improves or beneficially alters the symptoms of individuals suspected of having or who have developed vascular smooth muscle cell proliferative disease by administering the pharmaceutical composition.

[0023] The pharmaceutical compositions 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 their activity and / or expression.

[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] Furthermore, the pharmaceutical composition may further include the form of a complex with various nucleic acid transporters (viral or nonviral transporters) known in the art in order to increase the in vivo delivery efficiency of the miRNA inhibitor. Specifically, as viral transporters, lentiviruses, adenoviruses, and adeno-related viruses are used to deliver vectors encoding miRNA to the intracellular nucleus and express the miRNA inhibitor. As nonviral transporters, inorganic nanoparticle-based transporters whose size and shape can be adjusted using inorganic substances such as gold, carbon, and silica; polymer-based transporters such as PLGA and PEI that are particularly deformable for effective delivery in specific cells; and lipid transporters that utilize liposomes, which are formed of lipid bilayers and have a water-based internal phase that can encapsulate nucleic acids and other substances for delivery. These can protect the miRNA inhibitor and improve its stability during blood circulation.

[0026] The aforementioned 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. Furthermore, it can be administered as a single or multiple doses. It is important to administer the amount that provides the maximum effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0027] Furthermore, the pharmaceutical composition can be administered parenterally by the intended method (for example, intravenously, subcutaneously, intraperitoneally, or topically), and the dosage will vary depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0028] In one embodiment of the present invention, we identified upregulated miRNAs that regulate vascular smooth muscle cell function in vitro. The results showed that miR-132-3p and miR-370-3p had a significant inhibitory effect on vascular smooth muscle cell proliferation, and that miR-130b-5p, miR-132-3p, and miR-410-3p significantly suppressed monocyte adhesion to vascular smooth muscle cells. Through in-depth investigation of proliferation-related miR-132-3p and miR-370-3p, we confirmed that they may regulate phenotypic transduction and proliferation signals, respectively (Figure 2).

[0029] Furthermore, 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. The results showed that the expression of miR-132-3p and miR-370-3p was significantly increased in balloon-injured carotid arteries compared to the sham control group, and miR-130b-5p and miR-410-3p were substantially induced by balloon injury. The inhibitors of these miRNAs significantly reduced neointimal proliferation in balloon-injured lesions compared to the control group, and the inhibitor of miR-130b-5p promoted the recovery of a monolayer called reendothelialization. This confirmed that these four miRNAs play a clear role in arterial homeostasis, such as intravascular inflammation and proliferation (Figure 3).

[0030] Furthermore, in one embodiment of the present invention, in order to identify the core target genes of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p, we selected DEGs that were commonly predicted in rats and humans through a target prediction database using genes (DEGs) that were differentially expressed in the injured artery at two time points as target genes for each miRNA. By confirming that these target genes were downregulated in the injured artery, we revealed that the core target genes of miR-132-3p, miR-370-3p, miR-130b-5p, and miR-410-3p are SOCS2 (Suppressor of Cytokine Signalling 2), BMP7 (Bone morphogenetic protein 7), TSPAN2 (Tetraspanin-2), and SMAD6 (SMAD family member 6) (Figure 4).

[0031] Furthermore, in one embodiment of the present invention, miR-370 was detected in the blood of patients with unstable angina, type 2 diabetes, and hyperlipidemia. As a result of investigating the biological importance of the miR-370 / BMP (bone morphogenic protein)-7 axis in smooth muscle cell proliferation, high expression of miR-370-3p was confirmed in tubular tissue sections of human patients with type II and type IV arteriosclerosis lesions. Therefore, it was confirmed that miR-370 is a novel miRNA associated with atherosclerosis, and that miR-370 suppression increases the level of BMP7 protein in human vascular smooth muscle cells. BMP7 treatment significantly induces SMAD1 / 5 / 9 phosphorylation in human smooth muscle cells, and BMP7 depletion interferes with the suppression of smooth muscle cell proliferation by miR-370 inhibitors. Thus, it was confirmed that miR-370-dependent BMP7 expression is a prerequisite for smooth muscle cell growth and neointimal proliferation occurring in damaged 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 incorporating the miRNA inhibitors into vascular smooth muscle cells will be used to prevent or treat vascular smooth muscle cell proliferative disorders.

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

[0034] In this application, the term "diagnosis" means the confirmation of the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis can be interpreted as the act of objectively determining whether there has been excessive proliferation or migration of vascular smooth muscle cells in a target patient with respect to vascular smooth muscle cell proliferative disorders.

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

[0036] The aforementioned kit can be specifically selected from the group consisting of microarrays, aptamer chip kits, ELISA (enzyme-linked immunosorbent assay) kits, blotting kits, immunoprecipitation kits, immunofluorescence test kits, protein chip kits, RT-PCR kits, and combinations thereof, and more specifically, it may be an RT-PCT 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 aforementioned objectives, the present invention provides a method for providing information on the diagnosis of vascular smooth muscle cell proliferative disorders, comprising the steps of: a) measuring the expression level of miRNA in an isolated biological sample; and b) determining that an increase in the expression level of miRNA in a control group indicates a risk of developing vascular smooth muscle cell proliferative disorders.

[0038] The aforementioned "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 levels of miR-132-3p, miR-370-3p, miR-130b-5p, or miR-410-3p, and more specifically, measuring the expression level of miR-370-3p.

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

[0040] The invention may also include formulations for measuring the expression level of the miRNA or its target gene. The formulation for measuring the expression level means a formulation that specifically binds to and enables recognition of the miRNA or its target gene, or amplifies it. Specific examples include antibodies, primers, or probes that specifically bind to the miRNA or its target gene, but are not limited to these, and those skilled in the art should be able to select an appropriate formulation to suit the purpose of the invention.

[0041] The formulation can be directly or indirectly labeled for measuring the expression level of the miRNA or its target gene. Specifically, the labels can be, but are not limited to, ligands, beads, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent substances, chemiluminescent substances, magnetic particles, haptens, and dyes. Specific examples include, but are not limited to, biotin, avidin, and streptavidin as ligands, luciferase, peroxidase, and beta-galactosidase as enzymes, and fluorescein, coumarin, rhodamine, phycoerythrin, and sulforhodamate chloride (Texas Red) as fluorescent substances. Most known labels can be used as such detectable labels, and those skilled in the art should be able to select an appropriate label to suit the purpose of the invention.

[0042] The term "primer" refers to a short sequence having a short free 3' hydroxyl group that can form a base pair with a complementary template and functions as a starting point for copying the template strand. In the present invention, the primer used for miRNA amplification may be a single-stranded oligonucleotide that can act as a starting point for template-instructing DNA synthesis under appropriate conditions in a suitable buffer (e.g., four other nucleoside triphodes and a polymerization agent such as DNA, RNA polymerase, or reverse transcriptase) and at an appropriate temperature, although the appropriate length of the primer may vary depending on the intended use. The primer sequence does not need to be perfectly complementary to the polynucleotide of the miRNA of the gene or its complementary polynucleotide; it is acceptable 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 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 type II and type IV arteriosclerosis lesions, and the tubular tissue more specifically means, but is not limited to, tissue scraped from a blocked arterial vessel.

[0045] miR-370-3p has been detected in the blood of patients with unstable angina, type 2 diabetes, and hyperlipidemia. High expression of miR-370-3p in tubular tissue sections of patients with arteriosclerosis confirmed that it is a novel miRNA associated with atherosclerosis.

[0046] The method for providing information necessary to determine whether or not the disease described above has developed according to the present invention may include the step of quantitatively analyzing the expression level of miRNA or its target gene from the blood or tubular tissue of an individual suspected of having a vascular smooth muscle cell proliferative disorder, specifically atherosclerosis.

[0047] The aforementioned term, “individual,” may include, without limitation, mammals such as rats, livestock, and humans that are susceptible to or have developed vascular smooth muscle cell proliferative disorder. In another aspect of the present invention, in order to achieve the aforementioned objectives, the present invention can provide a formulation for measuring the expression level of miRNA or a miRNA target gene for producing a diagnostic formulation for vascular smooth muscle cell proliferative disorders.

[0048] The terms "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 aforementioned objectives, the present invention can provide a method for treating vascular smooth muscle cell proliferative disorders, comprising administering a pharmaceutical composition for the prevention or treatment of vascular smooth muscle cell proliferative disorders, comprising the miRNA expression inhibitor and a pharmaceutically acceptable carrier, to an individual in need thereof.

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

[0050] In this application, the term "individual" means any animal, including humans, that possesses or has developed the vascular smooth muscle cell proliferative disease of the present invention. By administering the pharmaceutical composition of the present invention to an individual, preventive and therapeutic effects of the said disease can be obtained.

[0051] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In this application, the term "administration" means introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the administration route can be through a variety of parenteral routes, as long as it can reach the target tissue.

[0052] The aforementioned pharmaceutical composition can be appropriately administered to an individual according to the usual methods, routes of administration, and dosages used in the art, as needed or for the purpose. Examples of routes of administration include parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Furthermore, appropriate dosages and frequency of administration can be selected by methods known in the art, and the actual amount and frequency of administration of the pharmaceutical composition of the present invention can be appropriately determined by a variety of factors such as the type of symptoms to be treated, route of administration, sex, health status, diet, age and weight of the individual, and severity of the disease.

[0053] In this invention, the term "pharmaceutically effective amount" means an amount sufficient to suppress or mitigate the increase in vascular permeability with a reasonable benefit / risk ratio applicable to medical use. The level of the effective dose can be determined by factors including the individual's species and severity, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, drugs used concurrently, and other factors well known in the medical field. For example, the dose can be 0.01 to 500 mg / kg per day, specifically 10 to 100 mg / kg, and the dose can be administered once or in several divided doses per day.

[0054] The compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. They can also be administered as single or multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0055] The compositions of the present invention can be used alone for the prevention or treatment of vascular smooth muscle cell proliferative disorders, or 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 aforementioned objectives, the present invention provides a method for screening miRNA target genes that suppress vascular smooth muscle cell proliferation or migration, which includes the step of profiling differentially expressed genes (DEGs) in injured arteries on days 3 and 5 after injury.

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

[0058] The profiling described in step 1) above refers to gene expression profiling, which allows for the simultaneous comparison of the expression levels of various genes between two or more samples. Northern blotting, microarray analysis, RNA sequencing, etc., can be used to analyze gene expression. In this invention, overall gene expression changes were confirmed through RNA sequencing, but the invention is not limited to this.

[0059] The target prediction database described in step 2) above is for predicting the target genes of miRNAs. The function of miRNAs is generally understood through the genes they regulate. As of October 2018, the database contains 38,589 human miRNAs (mature miRNAs) (miRBase database, version 22.1). Predictions can be made by aligning the 3'UTR of the target gene with the miRNA seed. Prediction accuracy is improved by adding structural features and evolutionarily conserved binding sites. Programs that provide such prediction results include DIANA-microT, MicroInspector, MiRanda, PicTar, RNA22, RNAhybrid, TargetBoost, TargetScan, miRDB, and miRmap. In this invention, TargetScan, miRDB, and miRmap were used, but the invention is not limited to these.

[0060] The target genes in step 3) above specifically refer to genes whose intracellular expression can be regulated by miRNA through complementary binding to mRNA, and there are many target genes for a single miRNA.

[0061] The screening in step 4) above refers to gene screening, which means selecting target genes present in the library. Screening methods include screening using hybridization, screening using antibodies, screening using differences in gene expression, and screening using binding to specific proteins. In this invention, screening using differences in gene expression was used, but the invention is not limited to this. [Examples]

[0062] The present invention will be described in more detail below through the examples provided. These examples are provided to illustrate the present invention more concretely, and the scope of the present invention is not limited by these examples.

[0063] Experimental Example 1: Fabrication of a carotid artery balloon injury model A carotid artery balloon injury model was created using 10-week-old male albino rats (Sprague-Dawley rats) that had been reared and then allowed to adapt to the environment for one week.

[0064] Specifically, balloon injury was induced in the left common carotid artery of rats using a Fogarty balloon embolectomy catheter. After exposing the left external carotid artery and electrocoagulating the surrounding arterial branches, the catheter was inserted through a transverse incision in the external carotid artery, positioned 1 cm medial to the cut site, inflated, and then moved back and forth along the common carotid artery to injure it. For catheter-mediated intra-arterial delivery of a miRNA inhibitor, a transfection complex (200 nM miRNA / 10 μl Lipofectamin RNAimax in 200 μl Opti-MEM) was injected into the injured carotid artery lumen and cultured for 15 minutes. Before removing the catheter, the lumen was rinsed once with saline, and after catheter removal, the perforated site was sealed, and the clap of the common carotid artery was released to re-establish blood flow. Unless otherwise specified, rats recovered in cages for up to 14 days thereafter.

[0065] Experimental Example 2. Histopathological Analysis Balloon-injured rats were anesthetized and fixed transcardiac perfusion-fixation with heparinized saline containing 3.7% formaldehyde, after which the common carotid artery was resected. The vessel was paraffin-embedded and cut with a rotary microtome (Leica RM2255). Two serial tissue sections (4 μm thick) were obtained from the middle portion of the common carotid artery and stained with haematoxylin and eosin. The lumen, internal lamina, and external lamina regions were measured using NIH Image v 1.62. The intima and medial regions were determined by subtracting the lumen region from the internal lamina region and the internal lamina region from the external lamina region, respectively. For analysis, the average of the values ​​obtained from two serial sections per rat was calculated.

[0066] Experimental Example 3: RNA Sequencing To perform small RNA and mRNA sequencing, total RNA was isolated from rat carotid artery tissue 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 an RIN value greater than 8 were used in the sequencing process.

[0067] First, a small RNA library was prepared for small RNA sequencing using the TruSeq Small RNA Prep kit (Illumina). Simply put, 1 μg of total RNA was ligated with 3' and 5' RNA adapters. Reverse transcription PCR was performed using primers that annealed to the 3' and 5' adapters to generate and enrich cDNA constructs. The cDNA library was purified using the Pippin prep electrophoresis platform (Sage Science), and library quality was validated with the 2100 Bioanalyzer (Agilent Technologies). Small RNA sequencing was performed using the Hiseq 2500 system (Illumina) at a 1X51 setting.

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

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

[0070] mirVana miRNA mimetic or inhibitor (Invitrogen, USA) and siRNA (Bioneer, Korea) were transfected using lipofectamine RNAimax (Invitrogen).

[0071] Place cells in a 6-well plate at a rate of 1 × 10⁶ 5The cells were dispensed at a density of cells / well. After 24 hours, transfection complexes containing miRNA mimetics (0.1 nM each), miRNA inhibitors (10–150 nM each), or siRNAs (100 nM each) were added to the culture plates, and then replaced with fresh culture medium 24 hours after transfection.

[0072] Experimental Example 5: Real-time quantitative PCR Total RNA was isolated from human vascular smooth muscle cells and rat carotid artery tissue cultured 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). The expression levels of mature miRNAs were quantified by 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 was performed at 95°C for 10 minutes, followed by denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 60 seconds, and this cycle was repeated 40 times for amplification.

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

[0075] The thermal cycling conditions for mRNA were as follows: Initial denaturation was performed at 95°C for 15 minutes, followed by denaturation at 94°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, repeated 40 times 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 rapidly frozen with 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 minutes, the purified lysates were separated into 30 μg portions on denatured polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated overnight with primary antibodies in Tris-buffered saline (TBS) solution containing 0.05% Tween®-20 and 5% BSA at 4°C. Next, the membrane was incubated for 1 hour with an HRP-conjugated secondary antibody diluted 1:3000 in a 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: Analysis of Cell Proliferation and Cell Cycle 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, grown for the indicated time, and then cultured with WST-1 reagent (10 μl / well) at 37°C for 1 hour. The number of viable cells was estimated by measuring absorbance at 450 nm.

[0079] For cell cycle analysis, human vascular smooth muscle cells (1 × 10⁻⁶) 5 Cells were harvested after 48 hours of transfection. The cells were fixed, permeabilized with 70% ethanol overnight at -20°C, treated with 100 μg / ml RNase A at 37°C for 1 hour, and then stained with 10 μg / ml propidium iodide. Intracellular DNA content was measured using the 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 tissue staining, paraffin sections of balloon-injured carotid arteries were deparaffinized with xylene and rehydrated with ethanol. The rehydrated tissue sections were boiled for 20 minutes in a citrate-based antigen unmasking solution (Vector Laboratories) for antigen retrieval. For dual immunofluorescence, the tissue sections were blocked for 1 hour in PBS-T (0.3% Triton X-100 in PBS) containing 5% normal donkey serum. The samples were then incubated overnight at 4°C with FITC-conjugated anti-vWF antibody (1:50 dilution, Abcam). 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 hours.

[0081] F-actin staining was performed by fixing human vascular smooth muscle cells with 3.7% formaldehyde for 15 minutes and permeabilizing them with PBS-T at room temperature for 15 minutes. The cells were then labeled with Alexa Fluor 488-conjugated phalloidin (cat. no A12379, Invitrogen) at room temperature for 60 minutes.

[0082] TUNEL analysis was performed by incubating the fixed cells with a permeable solution (0.1% Triton X-100, 0.1% sodium citrate) at 4°C for 2 minutes, and then incubating them 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 fluorescence images were obtained using an LSM880 Airyscan confocal microscope (Carl Zeiss). Experimental Example 9: Transwell migration assay Chemotactic cell migration was measured using a 24-well transwell culture chamber (Costar; 8 mM pore size). The upper chamber was coated with gelatin B (1 mg / ml) and air-dried for 1 hour. Human vascular smooth muscle cells were transfected with a miRNA inhibitor for 24 hours, followed by serum deprivation for 18 hours, and then re-plated into the upper chamber at a density of 6,000 cells / chamber using basal medium. Complete medium was added to the lower chamber, and the transwell chamber was incubated at 37°C for 24 hours. Unmigrated cells were removed from the upper side of the membrane, and cells that passed through and adhered to the lower side of the membrane were fixed and stained with 0.6% hematoxylin and 0.5% eosin. The number of stained cells was counted in four sections and the average was calculated.

[0084] Experimental Example 10: Monocyte adhesion assay Human vascular smooth muscle cells were transfected with a miRNA inhibitor for 24 hours and then plated again in 96-well plates (4,000 cells / well). Subsequently, the human vascular smooth muscle cells were stimulated with TNF-α (10 ng / ml) for 18 hours. Separately, monocyte U937 cells (1 × 10⁶) were transfected. 6 U937 cells (1 × 10⁶ cells / well) were stimulated with IFN-γ (50 μg / ml) for 24 hours. 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 × 10⁶ cells / well) were then analyzed. 5 The U937 cells (cell / well) were added to confluent human vascular smooth muscle cells and incubated at 37°C for 1 hour. Unbound U937 cells were smoothly removed by washing three times with PBS, and the attached U937 cells were detected and counted using a ZOE Fluorescent Cell Imager (Bio-Rad).

[0085] Experimental Example 11: Luciferase Reporter Analysis To predict the target gene of miR-370-3p, we purchased the reporter plasmid pMirTarget, containing the full-length 3'UTR sequence of the human BMP7 gene (hBMP7-3'UTR), from Origene (cat.No.SC218118). A negative control group was created by mutating two consecutive nucleotides within the miR-370-3p target region #1 (nucleotides 229-235) of the hBMP7-3'UTR. For reporter analysis, HEK293T cells were plated in 24-well plates and transfected with the reporter plasmid for 6 hours. Subsequently, these cells were transfected with the miR-370-3p mimetic for 48 hours, lysed, and subjected to protein analysis. The same amount of cell lysate was subjected to luciferase analysis. Luminescence signals were measured using a VICTOR Multilabel Plate Reader (Perkin Elmer).

[0086] Experimental Example 12: In situ hybridization miR-370-3p expression in arterial tissue was detected using the 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 at 37°C for 10 minutes, and hybridization was performed with miRNA detection probes (40 nM each) incubated at a temperature 30°C lower than the probe's RNA Tm value for 1 hour. The slides were washed with serial dilutions of saline-sodium citrate (SSC) hybridization buffer at each annealing temperature. Immunodetection was performed by incubating alkaline phosphatase-conjugated anti-DIG antibody (cat. No. 11093274910, Roche) at room temperature for 60 minutes. The slides were then incubated at 37°C for 2 hours with NBT / BCIP (cat. No. 11697471001, Roche) substrate solution containing 0.2 nM Levamisol to develop color. Scramble probes annealed to serial tissue sections were used as controls, and cell nuclei were labeled with Nuclear Fast Red.

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

[0088] Example 1: Confirmation of differentially expressed miRNAs in damaged arteries Based on histological analysis of neointimal thickening based on 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, and identified miRNAs that signal the initiation of smooth muscle cell dedifferentiation and proliferation.

[0089] Specifically, whole RNA samples were prepared and separated into small RNAs and messenger RNAs through appropriate purification procedures, followed by purification. Both RNA pools were subjected to library composition 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 two time points compared to the sham control group (Figure 1A). Each of these miRNAs was examined not only for their association with the vascular system but also for their novelty, and 50 miRNAs were selected for quantitative validation by miRNA-specific real-time PCR. The results showed that 12 upregulated miRNAs and 6 downregulated miRNAs exhibited more than five times the changes in the injured arteries compared to the sham control group. One-fifth of the miRNAs were temporarily downregulated three days after injury, suggesting their potential as early-response miRNAs. By excluding some miRNAs that were not identified in human databases or whose seed sequences did not match between rats and humans, we identified miRNAs such as miR-221-3p, miR-21-5p, and miR-146a-5p among the remaining candidate miRNAs as being associated with leiomyocyte hyperplasia {Sun, 2011 #2627; Liu, 2009 #2623; Ji, 2007 #2625}, thus supporting the validity of the selection strategy using a rodent model.

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

[0092] In this way, we discovered a subset of miRNAs, including some known candidates related to neointimal hyperplasia, through miRNA screening using a rodent model. Example 2. Upregulated miRNAs that regulate in vitro smooth muscle cell function To determine the role of upregulated miRNAs in smooth muscle cell biology, we performed in vitro analysis of three types of cellular basis 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, an indicator of inflammation, was markedly suppressed by three miRNAs: miR-130b-5p, miR-132-3p, and miR-410-3p. Based on these cell analyses, we conducted an in-depth investigation into proliferation-related miRNAs, namely miR-132-3p and miR-370-3p. Both miRNA inhibitors induced cell cycle arrest at the G1 stage, sustainably suppressing the time-dependent proliferation of human vascular smooth muscle cells (Figures 2B and 2C). Consistently, the levels of the cyclin-dependent kinase inhibitors p21 and p27 were significantly increased by both miRNA inhibitors (Figure 2D). However, this miRNA suppression did not result in apoptosis in human vascular smooth muscle cells, suggesting cell proliferation suppression of smooth muscle cells.

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

[0095] As the simplest method to mimic the in vivo arterial environment, human vascular smooth muscle cells were cultured at a high confluence point on laminin-coated plates to induce a contractile phenotype. When smooth muscle cells from the high confluence point were replated at a low confluence point, the cells were gradually transformed into a synthetic phenotype. Immunoblot analysis of Experimental Example 6 confirmed that transfection with the miR132-3p inhibitor restored the SMA level that had disappeared at the low confluence point (Figure 2E). Indeed, immunofluorescence staining of F-actin filaments demonstrated that the synthetic phenotype of human vascular smooth muscle cells at the low confluence point was transformed into a contractile phenotype by the miR-132-3p inhibitor (Figure 2F).

[0096] Therefore, we confirmed that miR-132-3p and miR-370-3p may modulate phenotypic transition and proliferation signals, respectively. Example 3. Upregulated miRNAs that regulate in vivo neointebral proliferation To evaluate the efficacy of in vivo miRNAs through a balloon injury model, the arterial expression levels of four functional miRNAs were investigated via in situ hybridization, as described in Experimental Example 12.

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

[0098] Catheter-mediated local intramural transmission of miRNA inhibitors significantly reduced neointimal proliferation in balloon-injured lesions compared to the control group (Figure 3B). Immunofluorescence staining of the carotid artery for von Willebrand (vWF) and SMA as EC and smooth muscle cell markers showed that miR-130b-5p inhibitors promoted the recovery of the EC monolayer, known as re-endothelialization (Figure 3C). Unlike smooth muscle cells, miR-130b-5p performed an antiproliferative function in the EC.

[0099] Through this, we confirmed that the four miRNAs mentioned above play a significant role in arterial homeostasis, such as intravascular inflammation and proliferation. Example 4. Identification of target genes of functional miRNAs Next, in order to understand the biological consequences of the miRNAs selected in Example 3, mRNA sequencing was performed, and differentially expressed genes (DEGs) were profiled in the carotid arteries of balloon-damaged rats.

[0100] As a result, as shown in Figure 4, we confirmed that 3,699 DEGs were significantly modified at 3 and 5 days post-injury compared to the sham control group (Figure 4a). Heatmap analysis showed that there were roughly equal numbers of upregulated and downregulated DEGs (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). DEGs that showed consistently high prediction scores for both rats and humans in one or more databases (TargetScan < -0.1, miRDB > 70, miRmap > 70) were selected as predicted targets for each miRNA. Gene ontology (GO) enrichment analysis further identified a pool of functionally associated target genes with cognate miRNAs, and then, via real-time PCR, identified genes that were downregulated in balloon-injured carotid arteries compared to the sham control group (Figure 4c). For human relevance, the expression of the aforementioned representative genes was further investigated in human vascular smooth muscle cells transfected with miRNA inhibitors.

[0101] As a result, we identified the core target genes for each miRNA via real-time PCR and confirmed that SOCS2, BMP7, TSPAN2, and SMAD6 are specific targets for 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, since 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 tissue to investigate the biological importance 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 type II and IV atherosclerosis lesions via in situ hybridization (Figure 5a), confirming that miR-370 is a novel miRNA associated with atherosclerosis. Subsequently, the relationship between miR-370 and BMP7 was investigated in cultured human vascular smooth muscle cells, and indeed, serum stimulation induced miR-370-3p expression but conversely decreased BMP7 expression (Figure 5b). Consistently, reporter analysis using the 3-UTR region of the human BMP7 gene showed that the expression of luciferase containing a wild-type UTR (not a mutant UTR) was indeed reduced by the miR-370 mimetic (Figure 5d). Furthermore, Western blot analysis clearly showed that miR-370 suppression increased the level of BMP7 protein in human smooth muscle cells (Figure 5d).

[0104] These results collectively confirm that the BMP7 gene is a true miR-370 target in human smooth muscle cells. Since treatment with recombinant BMP7 has been 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 significantly induced SMAD1 / 5 / 9 phosphorylation in human vascular smooth muscle cells (Figure 5E), which suggests the anti-mitotic effect of BMP7 in smooth muscle cells. Furthermore, cell proliferation analysis demonstrated that BMP7 depletion restored smooth muscle cell proliferation that had been 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 damaged 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-inhibiting effect on smooth muscle cells. Through this, it can be predicted that incorporating the miRNA inhibitors into smooth muscle cells will be effective in preventing or treating vascular smooth muscle cell proliferative disorders.

[0106] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A pharmaceutical composition for the prevention or treatment of vascular smooth muscle cell proliferative disorders, comprising a miRNA (microRNA) inhibitor as an active ingredient, The aforementioned miRNA is miR-132-3p, and The aforementioned miRNA inhibitor is a pharmaceutical composition selected from the group consisting of siRNA, aptamers, or antisense oligonucleotides that are specific to miRNA.

2. The pharmaceutical composition according to claim 1, wherein the miRNA inhibitor regulates in vitro vascular smooth muscle cell function and in vivo neointimal proliferation by suppressing miRNAs upexpressed in damaged arteries.

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

4. A method for treating vascular smooth muscle cell proliferative disorders, comprising administering a pharmaceutical composition for the prevention or treatment of vascular smooth muscle cell proliferative disorders, comprising a miRNA expression inhibitor and a pharmaceutically acceptable carrier, to individuals other than those requiring it, The aforementioned miRNA is miR-132-3p, and The treatment method wherein the miRNA expression inhibitor is one selected from the group consisting of miRNA-specific siRNA, aptamers, or antisense oligonucleotides.

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