KR102531265B1 - Composition for preventing or treating valvular heart diseases comprising RSPO3 inhbitors - Google Patents

Abstract

The present invention relates to a composition for preventing or treating valvular heart disease comprising an RSPO3 expression or activity inhibitor as an active ingredient, wherein the RSPO3 expression or activity inhibitor can inhibit calcification of blood vessels or valves. Therefore, it can be usefully used for the prevention or treatment of related diseases.

Description

Composition for preventing or treating valvular heart diseases comprising RSPO3 inhibitors

The present invention relates to a composition for preventing or treating heart valve disease comprising an RSPO3 expression or activity inhibitor as an active ingredient.

Heart valve disease is a disease in which the opening and closing action of the valve is not good. When the valve does not open well functionally, it is called stenosis. This case is called insufficiency/regurgitation. Since there are four chambers in the human heart, there are four valves, and the most clinically problematic are the aortic valve and the two mitral valves.

Calcific aortic valve disease is a disease caused by calcification of the aortic valve. It is one of the most common heart valve diseases, and the incidence of the disease is increasing as the elderly population increases. . Calcific aortic valve disease may progress to aortic valve sclerosis and then progress to aortic valve stenosis.

The fundamental treatment of heart valve disease is valve replacement surgery in which the diseased valve is removed and replaced with an artificial valve. However, it is necessary to develop a material capable of suppressing calcification of the aortic valve in that the risk of the operation itself and problems that may occur after the operation should be considered.

KR 10-1598612 B

An object of the present invention is to provide a pharmaceutical composition for preventing or treating vascular or valve calcification, comprising an inhibitor of expression or activity of RSPO3 (R-spondin 3) as an active ingredient.

Another object of the present invention is 1) administering a candidate substance to a subject in whom vascular or valve calcification is induced;

2) isolating a biological sample from the subject to which the candidate substance is administered;

3) comparing the expression of the RSPO3 gene or protein with a control group from the separated biological sample; and

4) in step 3), confirming a decrease in expression of the RSPO3 gene or protein compared to the control group; to provide a screening method for a candidate substance for preventing or treating vascular or valve calcification, including

The present invention provides a pharmaceutical composition for preventing or treating vascular or valve calcification comprising an inhibitor of the expression or activity of RSPO3 (R-spondin 3) as an active ingredient.

The present invention comprises the steps of: 1) administering a candidate substance to a subject in whom vascular or valve calcification is induced;

2) isolating a biological sample from the subject to which the candidate substance is administered;

3) comparing the expression of the RSPO3 gene or protein with a control group from the separated biological sample; and

4) in step 3), confirming a decrease in the expression of the RSPO3 gene or protein compared to the control group;

The present invention relates to a composition for preventing or treating valvular heart disease comprising an RSPO3 expression or activity inhibitor as an active ingredient, wherein the RSPO3 expression or activity inhibitor can inhibit calcification of blood vessels or valves. Therefore, it can be usefully used for the prevention or treatment of related diseases.

1 shows a normal valve-derived VIC cell group (control); VIC cells derived from valves with calcified lesion formation (CAVD) were not treated with Pro-calcific medium (patient(-) group); Or, it shows the RNA sequencing analysis method and its results in the treatment group (patient (+) group).
Figure 2 shows a normal valve-derived VIC cell group; and GO (gene ontology) analysis results in a group in which pro-calcific medium was treated in valve-derived VIC cells with calcified lesion formation.
Figure 3 shows the results of measuring the expression levels of BMP2, RUNX2, and RSPO3 through real-time PCR after treating normal valve-derived VIC cells and valve-derived VIC cells with calcified lesion formation with Pro-calcific medium, respectively. (AR(-): aortic regurgitation (control) without pro-calcific medium; AR(+): aortic regurgitation (control) with pro-calcific medium; AS(-): aortic stenosis (patient) without pro-calcific medium; and AS(+): aortic stenosis (patient) with pro-calcific medium).
4 shows the results of Alizarin Red S staining and immunohistochemistry for RSPO3 on normal valve tissue (Non-CAVD) and valve tissue (CAVD) of patients with calcific aortic valve disease.
5 shows the results of immunohistochemistry for RSPO3 in normal valve tissue (Non-CAVD) and valve tissue (CAVD) of patients with calcific aortic valve disease.
Fig. 6A shows a group treated with control siRNA to valve-derived VIC cells with calcified lesion formation (siControl Cal-); A group treated with pro-calcific medium and control siRNA to valve-derived VIC cells with calcified lesion formation (siControl Cal+); And the results of measuring the expression levels of BMP2 and RSPO3 through real-time PCR for the group (siRSPO3 Cal+) treated with Pro-calcific medium and treated with RSPO3 siRNA to valve-derived VIC cells with calcified lesion formation.
Figure 6B is a group (siControl) treated with pro-calcific medium and control siRNA to valve-derived VIC cells with calcified lesion formation; and the results of Alizarin Red S staining for the group (siRSPO3) treated with Pro-calcific medium and treated with RSPO3 siRNA to valve-derived VIC cells with calcified lesion formation.

The present invention provides a pharmaceutical composition for preventing or treating vascular or valve calcification comprising an inhibitor of the expression or activity of RSPO3 (R-spondin 3) as an active ingredient.

In the present invention, "vascular or valve calcification" means the formation, growth or deposition of extracellular matrix hydroxyapatite (calcium phosphate) crystal deposits in blood vessels or valves.

The vascular calcification includes calcification of coronary artery, aorta and other blood vessels, and may be, for example, medial calcification or atherosclerotic calcification.

The medial calcification may be used as the same meaning as medial wall calcification or Moenckeberg's sclerosis, and refers to calcification caused by calcium deposited in the medial wall. It is known that such mesenchymal calcification is caused by the expression of bone formation-stimulating factors in blood vessels due to diabetes, abnormal calcium metabolism, or renal disease.

The atherosclerotic calcification refers to calcification occurring in atheromatous plaques along the intimal layer. It is known that such atherosclerotic calcification is caused by direct calcium deposition at the site of an atherosclerotic lesion.

The vascular or valve calcification can be induced in the human heart, lungs, kidneys and aorta, but is not limited to all tissues and organs in which calcification can occur.

When the calcification occurs in the heart, heart valve disease is caused, which is a disease in which the opening and closing action of the valve is not good, and when the valve does not open well functionally, it is called stenosis. When blood does not close tightly, it is called insufficiency/regurgitation.

The heart valve disease includes calcific aortic valve disease (CAVD), aortic stenosis, mitral stenosis, aortic regurgitation and mitral valve regurgitation. regurgitation), preferably calcific aortic valve disease or aortic valve stenosis, but is not limited thereto.

The calcific aortic valve disease or aortic valve stenosis may be caused by calcification of the aortic valve.

The RSPO3 may consist of the amino acid sequence of SEQ ID NO: 1, and the gene encoding the RSPO3 may consist of the nucleotide sequence of SEQ ID NO: 2.

The RSPO3 expression or activity inhibitor may be an RSPO3 gene expression inhibitor or an RSPO3 protein expression or activity inhibitor. The RSPO3 gene expression inhibitor is selected from the group consisting of an antisense oligonucleotide, siRNA (small interfering RNA), shRNA (short hairpin RNA) and aptamer that bind complementary to the RSPO3 gene mRNA It may be any one, but is not limited thereto.

The RSPO3 protein expression or activity inhibitor is any compound selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the RSPO3 protein. It may be one, but is not limited thereto.

The "antisense oligonucleotide" is DNA, RNA, or a derivative thereof containing a nucleic acid sequence complementary to the mRNA nucleotide sequence of a target gene, and inhibits translation of mRNA into protein by binding to a complementary sequence in mRNA. do.

A double consisting of a sense sequence and an antisense sequence with a length of 15 to 30 bp capable of suppressing the expression of the target gene through RNAi (RNA interference) by mRNA cleavage by complementarily binding to the mRNA nucleotide sequence of the "siRNA" target gene As -stranded RNA, it can be used for gene knockdown or gene therapy.

In the present invention, the siRNA is an RNA molecule of a sense sequence (UGUGAUACCUGUUUCAACAtt) consisting of the nucleotide sequence of SEQ ID NO: 3 and an antisense sequence (UGUUGAAACAGGUAUCACAgt) consisting of the nucleotide sequence of SEQ ID NO: 4 capable of complementarily binding to the mRNA of the RSPO3 gene. it could be

The "shRNA" is an RNA having a hairpin structure capable of silencing the expression of a target gene through RNAi (RNA interference) by complementarily binding to the mRNA nucleotide sequence of the target gene. After being cleaved into siRNA, it binds to RNA-induced silencing complex (RISC), and RISC can be used to suppress gene expression by binding to mRNA and cleaving it.

The "peptide mimic" refers to a peptide or non-peptide that inhibits the activity of a target protein by inhibiting the binding domain of the target protein.

The "aptamer" is an oligonucleotide or peptide molecule that specifically binds to a target molecule, and in the case of a nucleic acid aptamer, an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment) It is possible to isolate and obtain oligomers that bind to specific chemical or biological molecules with high affinity and selectivity. An aptamer can specifically bind to a target and modulate the activity of the target, eg blocking the target's function through binding.

The "antibody" refers to a substance that reacts to the invasion of an antigen, which is an external substance while circulating in the blood or lymph in the body's immune system, and is also called immunoglobulin as a globulin-based protein formed in lymphoid tissue. Antibodies are produced by B cells and bind specifically to antigens. One antibody molecule has two heavy chains and two light chains, and each heavy chain and light chain each include a variable region at the N-terminus. Each variable region is composed of 3 complementarity determining regions (CDRs) and 4 framework regions (FRs). It exists as a relatively short peptide sequence maintained as formation sites.

Such antibodies include polyclonal antibodies, monoclonal antibodies, recombinant antibodies and complete forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, such as Fab, F(ab' ), F(ab')2 and Fv.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means that it exhibits non-toxic properties to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like.

In addition, the pharmaceutical composition of the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Furthermore, it may be used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or skin external preparations in the form of gels. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance into a subject by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, but not limited thereto.

The term "individual" refers to all animals such as rats, mice, livestock, and the like, including humans. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's gender, age, Factors including body weight, health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, drugs used in combination or concurrently, and other medical disciplines. It can be easily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dose, or may be administered in several divided doses.

The present invention comprises the steps of: 1) administering a candidate substance to a subject in whom vascular or valve calcification is induced;

2) isolating a biological sample from the subject to which the candidate substance is administered;

3) comparing the expression of the RSPO3 gene or protein with a control group from the separated biological sample; and

4) in step 3), confirming a decrease in the expression of the RSPO3 gene or protein compared to the control group;

The method may further include a step of selecting a candidate material having a reduced expression level of RSPO3 protein or a gene encoding the same than a reference value.

The measurement is RT-PCR (reverse transcription polymerase chain reaction), competitive RT-PCR, real-time quantitative RT-PCR, RNase protection method, northern blot, DNA chip technology assay), enzyme-linked immunosorbent assay (ELISA), western blot, immunoblot, or immunocytochemistry.

The present invention provides a diagnostic composition for heart valve disease (valvular heart disease) comprising an agent capable of measuring the expression level of RSPO3 (R-spondin 3) protein or a gene encoding the same.

An agent for measuring the expression level of the protein may be an antibody, an interacting protein, a ligand, nanoparticles, or an aptamer that specifically binds to the protein or peptide fragment, but is not limited thereto. .

An agent for measuring the expression level of the gene encoding the protein may be a primer or probe that binds to the mRNA of the gene, but is not limited thereto.

As used herein, the term "primer" is a base sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and serving as a starting point for template strand copying. A short sequence of bases that acts Primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerate or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a gene, and is labeled so that the presence or absence and expression level of a specific gene can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. Selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. These nucleotide sequences can also be modified through various methods known in the art. Examples of such modifications are methylation, capping, substitution of one or more natural nucleotides with homologs, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or to charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.).

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. RNA sequencing analysis

The present inventors obtained normal valves without formation of calcified lesions and valves with formation of calcified lesions from patients who underwent aortic valve replacement, and after isolating and culturing valvular interstitial cell (VIC) cells from the valves, RNA sequencing analysis was performed, respectively. The gene expression profile of was analyzed. The experiment was conducted in a normal valve-derived VIC cell group (control group), a valve-derived VIC cell with calcified lesion formation, without pro-calcific medium treatment (patient(-) group)), or a treatment group (patient(+) group). (Fig. 1 upper panel). Each cell was cultured for 7 days and then used for analysis.

As a result, the gene expression profile of valve-derived VIC cells with calcific lesion formation in the non-treated or treated group with Pro-calcific medium was different compared to the control group, which was the normal valve-derived VIC cell group. In addition, it was confirmed that the gene expression profile was different between the non-treated or treated groups of pro-calcific medium in valve-derived VIC cells with calcified lesion formation (Fig. 1 lower panel).

From the above results, the present inventors confirmed that the expression of various genes is regulated according to the presence or absence of calcification valve disease and the induction of calcification.

In addition, according to the GO (gene ontology) analysis, the up-regulation groups include regulation of actin filament-based movement (GO:1903115), negative regulation of microtubule polymerization (GO:0031115), negative regulation of bone mineralization ( GO:0030502), followed by down-regulation of type I interferon signaling pathway (GO:0060337), protein localization to chromosome, and centromeric region (GO:0071459) (FIG. 2).

Among the up-regulated gene groups, the present inventors identified genes whose expression is increased in valve-derived VIC cells with calcified lesion formation compared to normal valve-derived VIC cells, which are control groups, and whose expression is further increased when calcification is induced, genes highly related to calcification. Determined to be , the rate of increase in expression of the gene was analyzed. As a result, RSPO3 (R-spondin 3) was up-regulated 3.16-fold in valve-derived VIC cells with calcified lesion formation compared to normal valve-derived VIC cells, and up-regulated 2.74-fold when calcification was induced, the most significantly increased expression. confirmed to be genetic.

Example 2. In valve-derived VIC cells Expression analysis of RSPO3 gene

The present inventors found that RSPO3 (R-spondin 3) was found to have higher expression levels of genes in valve-derived VIC cells with calcified lesion formation compared to BMP2 (bone morphogenetic protein 2) and RUNX2 (Runt-related transcription factor 2), known as calcification-related genes. An experiment was performed to verify whether or not it increases. Briefly, normal valve-derived VIC cells and valve-derived VIC cells with calcified lesions were treated with Pro-calcific medium, respectively, and then cultured for 4 and 7 days, and then gene expression levels were confirmed through real-time PCR. .

As a result, the expression level of the RSPO3 gene was found to be significantly up-regulated when calcification was induced by treating valve-derived VIC cells with calcified lesion formation with Pro-calcific medium, the same as the calcification-related genes BMP2 and RUNX2 ( Fig. 3).

From the above results, the present inventors verified that the RSPO3 gene was upregulated in valve-derived VIC cells with advanced calcification.

Example 3. RSPO3 expression analysis according to aortic valve calcification

The present inventors performed an experiment to compare the expression of RSPO3 according to the degree of calcification in human aortic valve tissue. Briefly, a human aortic valve sample isolated from a patient was fixed in a 4% formalin solution and then embedded in paraffin to prepare a 4 μm thick section. Alizarin Red S staining was performed to confirm the degree of calcification of the valve tissue. As a result, it was confirmed that calcification appeared significantly in the valves of patients with aortic valve stenosis (calcified aortic valve disease) compared to normal valves (FIG. 4).

In addition, the present inventors performed immunohistochemical staining (immunohistochemistry) to confirm the expression of RSPO3 in valve tissues. As a result, it was confirmed that the expression of RSPO3 was significantly higher in the valves of patients with aortic valve stenosis (calcified aortic valve disease) than in normal valves (FIGS. 4 and 5).

From the above results, the present inventors confirmed that the valves of patients with aortic valve stenosis (calcified aortic valve disease) were significantly calcified compared to normal valves, and that the expression of RSPO3 was remarkably high.

Example 4. Effect of RSPO3 knockdown on calcification

In order to confirm whether RSPO3 induces calcification, the present inventors performed an experiment to confirm the effect on calcification by knocking down RSPO3 in valve-derived VIC cells. Briefly, a group treated with control siRNA to valve-derived VIC cells with calcified lesion formation (siControl Cal-); A group treated with pro-calcific medium and control siRNA to valve-derived VIC cells with calcified lesion formation (siControl Cal+); And the expression levels of BMP2 and RSPO3 were measured by real-time PCR for the group (siRSPO3 Cal+) treated with Pro-calcific medium and RSPO3 siRNA to valve-derived VIC cells with calcified lesion formation.

As a result, when RSPO3 siRNA was treated, the expression of RSPO3 was significantly lowered, confirming that RSPO3 expression could be inhibited by siRNA treatment, and the expression of BMP2, a calcification-related gene, could also be inhibited by RSPO3 siRNA. (Fig. 6A).

In addition, the present inventors performed Alizarin Red S staining to compare the degree of calcification according to RSPO3 siRNA treatment. As a result, it was confirmed that treatment with RSPO3 siRNA (siRSPO3) significantly reduced calcification compared to treatment with control siRNA (FIG. 6B).

From the above results, the present inventors confirmed that calcification of the aortic valve can be inhibited by suppressing the expression of RSPO3. Accordingly, the present inventors found that RSPO3 expression or activity inhibitors can be used to prevent or treat heart valve diseases such as calcific aortic valve disease or aortic stenosis by inhibiting calcification of the aortic valve. confirmed that there is

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Composition for preventing or treating valvular heart diseases comprising RSPO3 inhbitors <130> PN2010-485 <150> KR 10-2019-0132769 <151> 2019-10-24 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 272 <212> PRT <213> artificial sequence <220> <223> RSPO3 protein <400> 1 Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met 1 5 10 15 Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg 20 25 30 Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40 45 Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala 50 55 60 Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys 65 70 75 80 Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95 Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr 100 105 110 Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn 115 120 125 Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser 130 135 140 Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr 145 150 155 160 Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val 165 170 175 Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro 180 185 190 Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys 195 200 205 Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn 210 215 220 Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser 225 230 235 240 Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys 245 250 255 Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His 260 265 270 <210> 2 <211> 819 <212> DNA <213> artificial sequence <220> <223> RSPO3 gene <400> 2 atgcacttgc gactgatttc ttggcttttt atcattttga actttatgga atacatcggc 60 agccaaaacg cctcccgggg aaggcgccag cgaagaatgc atcctaacgt tagtcaaggc 120 tgccaaggag gctgtgcaac atgctcagat tacaatggat gtttgtcatg taagcccaga 180 ctattttttg ctctggaaag aattggcatg aagcagattg gagtatgtct ctcttcatgt 240 ccaagtggat attatggaac tcgatatcca gatataaata agtgtacaaa atgcaaagct 300 gactgtgata cctgtttcaa caaaaatttc tgcacaaaat gtaaaagtgg attttactta 360 caccttggaa agtgccttga caattgccca gaagggttgg aagccaacaa ccatactatg 420 gagtgtgtca gtattgtgca ctgtgaggtc agtgaatgga atccttggag tccatgcacg 480 aagaagggaa aaacatgtgg cttcaaaaga gggactgaaa cacgggtccg agaaataata 540 cagcatcctt cagcaaaggg taacctgtgt cccccaacaa atgagacaag aaagtgtaca 600 gtgcaaagga agaagtgtca gaagggagaa cgaggaaaaa aaggaaggga gaggaaaaga 660 aaaaaaccta ataaaggaga aagtaaagaa gcaatacctg acagcaaaag tctggaatcc 720 agcaaagaaa tcccagagca acgagaaaac aaacagcagc agaagaagcg aaaagtccaa 780 gataaacaga aatcggtatc agtcagcact gtacactag 819 <210> 3 <211> 19 <212> RNA <213> artificial sequence <220> <223> RSPO3 siRNA sense <400> 3 19 <210> 4 <211> 19 <212> RNA <213> artificial sequence <220> <223> RSPO3 siRNA antisense <400> 4 uguugaaaca gguaucaca 19

Claims (15)

Selected from the group consisting of an antisense nucleotide complementary to mRNA of the RSPO3 (R-spondin 3) gene, small hairpin RNA (shRNA) and small interfering RNA (siRNA) A pharmaceutical composition for preventing or treating vascular or valve calcification, comprising an RSPO3 gene expression inhibitor as an active ingredient. According to claim 1,
The vascular or valve calcification is a pharmaceutical composition for the prevention or treatment of vascular or valve calcification, characterized in that caused by the human heart, lungs, kidneys and aorta. delete delete According to claim 1,
The vascular or valve calcification is the regulation of actin filament-based movement, negative regulation of microtubule polymerization, and negative regulation of bone mineralization. A pharmaceutical composition for preventing or treating vascular or valve calcification, characterized in that gene expression related to is up-regulated. According to claim 1,
The vascular or valve calcification is characterized by down-regulation of gene expression related to the type I interferon signaling pathway and proteins localized to chromosomes and centromeric regions; or A pharmaceutical composition for preventing or treating valve calcification. According to claim 1,
The RSPO3 is a pharmaceutical composition for preventing or treating vascular or valve calcification, characterized in that it comprises the amino acid sequence of SEQ ID NO: 1. According to claim 1,
The RSPO3 is a pharmaceutical composition for preventing or treating vascular or valve calcification, characterized in that it comprises the nucleotide sequence of SEQ ID NO: 2. delete delete According to claim 1,
The siRNA is a pharmaceutical composition for preventing or treating vascular or valve calcification, characterized in that siRNA comprising SEQ ID NO: 3 and SEQ ID NO: 4. delete 1) treating valve cells isolated from a subject with vascular or valve calcification with a candidate substance;
2) comparing the expression of the RSPO3 gene or protein with a control group in valve cells treated with the candidate substance; and
3) in step 2), confirming a decrease in the expression of the RSPO3 gene or protein compared to the control group; a method for screening candidates for preventing or treating vascular or valve calcification, including In the thirteenth,
Expression of the RSPO3 gene is selected from the group consisting of reverse transcription polymerase chain reaction, competitive polymerase chain reaction, real-time polymerase chain reaction, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, and DNA microarray method A method for screening candidates for the prevention or treatment of vascular or valve calcification, characterized in that carried out by the method. According to claim 13,
The expression of the RSPO3 protein was determined by Northern blot, Western blot, ELISA (Enzyme Linked Immuno Sorbent Assay), radiation immunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and mass spectrometry (Mass Spectrometry). A method for screening a candidate substance for preventing or treating vascular or valve calcification, characterized in that it is performed by a method selected from the group consisting of spectrometry and protein microarray methods.

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