CN118078996B - Use of RAB31 inhibitors in the preparation of products for preventing and/or treating cardiovascular diseases - Google Patents

Abstract

The present invention discloses the use of a RAB31 inhibitor in preparing a product for preventing and/or treating cardiovascular diseases. The present invention inhibits the expression of the RAB31 gene, can specifically inhibit and reduce myocardial cell inflammatory infiltration, reduce myocardial cell loss, inhibit myocardial cell apoptosis, inhibit myocardial fibrosis or treat ventricular remodeling after myocardial infarction, and the present invention opens up a new direction for the treatment of cardiovascular diseases, especially myocardial infarction.

Description

Use of RAB31 inhibitors for the preparation of products for the prevention and/or treatment of cardiovascular diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of an RAB31 inhibitor in preparation of a product for preventing and/or treating cardiovascular diseases.

Background

Cardiovascular disease is the first killer of human health. Ischemic heart disease is the leading cause of cardiovascular death, myocardial infarction (myocardial infarction, MI) is a common type of ischemic heart disease, and blocking of myocardial blood supply for more than 30 minutes can cause ultrastructural changes and dysfunction of various organelles, resulting in irreversible damage and even death of cardiomyocytes.

Acute Myocardial Infarction (AMI) is a critical clinical condition that severely affects human health. In recent years, the incidence rate of myocardial infarction has a remarkable rising trend in China, and the death rate has a rapid rising trend from 2005, which has become a serious public health problem. Ventricular Remodeling (VR) after acute myocardial infarction refers to the process of changing the size, shape and tissue structure of the ventricle and the morphology and structure of myocardial cells, and is an important cause of heart failure and death of patients. Therefore, inhibition of ventricular remodeling after myocardial infarction is an important approach to preventing and treating myocardial infarction. At present, a plurality of ventricular remodeling methods are adopted, and certain progress is made, but the treatment effect is still not ideal and the death rate is still higher due to the complex pathogenesis, side effects of medicines and the like. Therefore, it is urgent to find a safer and more effective control strategy.

Rab-related Protein 31 (RAB 31) belongs to the RAS superfamily and is a member of the small molecule GTPase family. It is localized intracellularly primarily to the trans-golgi network and endosomes, plays a role in regulating and controlling physiological processes such as endocytic exocytosis, vesicle transport, membrane targeting, fusion and the like of cells. RAB31 mediates the transport of vesicles from the golgi apparatus to early and late endosomes during the cell vesicle cycle. Several studies have reported that RAB31 plays a role in promoting tumor proliferation in malignant tumors. For example, RAB31 is strongly over-expressed in an estrogen receptor alpha positive breast cancer sample and promotes proliferation of breast cancer cells, RAB31 is one of important genes affected by development of glioblastoma multiforme and is up-regulated in glioblastoma expression, RAB31 is over-expressed in gastric cancer substances and interacts with GLI1 to play a role in oncogene in generation and development of gastric cancer, in addition, RAB31 is highly expressed in pancreatic cancer, is related to low survival rate of patients and is an effective prognosis biomarker in pancreatic cancer, RAB31 is highly expressed in cervical cancer cells and tissues, and expression of RAB31 is reduced can inhibit invasion and metastasis of cervical cancer cells, and over-expression of RAB31 promotes invasion and metastasis of cervical cancer cells.

In recent years, RAB31 has been found to promote endoluminal vesicle formation, inhibit degradation of the multivesicular endosome, and promote secretion of exosomes. RAB31 promotes endocytosis of TGF- β receptor II, activates TGF- β pathway, activates hepatic stellate cells, and causes liver fibrosis to occur.

However, the effect of RAB31 on myocardial infarction has not been reported.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of RAB31 inhibitors for the preparation of a product for the prevention and/or treatment of cardiovascular diseases, in order to solve the above-mentioned problems.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

The first aspect of the invention protects the use of a RAB31 inhibitor in the manufacture of a product for the prevention and/or treatment of cardiovascular diseases.

Preferably, the cardiovascular disease is ischemic heart disease. More preferably, myocardial infarction.

Preferably, the RAB31 inhibitor is a substance capable of inhibiting transcription or translation of the RAB31 gene, or capable of inhibiting expression or activity of the RAB31 protein.

More preferably, the RAB31 inhibitor is selected from one or more of a nucleic acid molecule, a small molecule chemical, an antibody, a polypeptide, a protein, a nucleic acid construct, a interfering lentivirus, an interfering adeno-associated virus, and a gene editing system.

Further preferred, the nucleic acid molecule is any one or more of an antisense oligonucleotide, double stranded RNA or shRNA.

Still further preferred, the RAB31 inhibitor is a nucleic acid molecule, the target sequence of which comprises the sequence shown in SEQ id No. 1. Preferably, the nucleic acid molecule comprises the element LoxP-target sequence-LoxP.

Further preferred, the RAB31 inhibitor is a nucleic acid construct comprising the sequence shown in SEQ ID No. 1.

Further preferred, the gene editing system is selected from one or more of the Cre-Loxp system, flp-FRT system, dre-ROX system, vCre-vloxp system, sCre-sloxp system, CRISPR/Cas9 system, CRISPR/Cas12 system, and/or CRISPR/Cas derived single base editing system.

Still more preferably, the RAB31 inhibitor is a gene editing system that targets the sequence shown in SEQ ID No. 1.

Preferably, the RAB31 inhibitor has the effect of preventing and/or treating myocardial infarction by at least one of:

Reducing inflammatory infiltration of myocardial cells;

reducing cardiomyocyte depletion;

Inhibiting cardiac hypofunction;

inhibiting myocardial fibrosis;

improving heart reconstruction after myocardial infarction.

Preferably, the RAB31 inhibitor is the only active ingredient or one of the active ingredients of the product.

In a second aspect the invention provides a nucleic acid molecule for reducing the expression of the RAB31 gene, the target sequence of which comprises the sequence shown in SEQ ID NO. 1.

A third aspect of the invention protects a substance associated with a nucleic acid molecule as hereinbefore described, said substance comprising one or more of:

1) A nucleic acid construct comprising a nucleic acid molecule as described above;

2) A gene editing system targeting a nucleic acid molecule as described above and knocked out, or comprising 1) a nucleic acid construct as described above;

3) A cell line comprising a nucleic acid molecule as described above, or comprising 1) a nucleic acid construct as described above, or obtained by transfecting a host cell with 2) the gene editing system;

4) A pharmaceutical composition comprising 1) said nucleic acid construct, or 2) said gene editing system, or 3) said cell line.

The fourth aspect of the invention protects the use of a nucleic acid molecule as described above or a substance as described above for the preparation of a product for the prevention or treatment of cardiovascular disease and/or for the preparation of a kit for reducing the expression of the RAB31 gene.

Preferably, the product for preventing or treating cardiovascular diseases has at least one of the following effects:

Reducing inflammatory infiltration of myocardial cells;

reducing cardiomyocyte depletion;

Inhibiting cardiac hypofunction;

inhibiting myocardial fibrosis;

improving ventricular remodeling after myocardial infarction.

In a fifth aspect, the present invention provides a method for screening a drug for preventing and/or treating cardiovascular diseases, which comprises targeting RAB31 as a drug, and searching for a substance capable of inhibiting or blocking the expression and/or function of RAB31 as a candidate drug.

The sixth aspect of the invention protects the use of the RAB31 gene as a target in the manufacture of a product for the prevention, treatment or diagnosis of cardiovascular disease.

Compared with the prior art, the invention has the following beneficial effects:

The invention discovers for the first time that RAB31 can be used as a potential target site to be applied to screening medicaments for preventing or treating cardiovascular diseases, in particular to screening medicaments for myocardial infarction. In addition, the expression of the RAB31 gene can be specifically inhibited and lightened, myocardial cell inflammation infiltration is reduced, myocardial cell deficiency is reduced, cardiac hypofunction is inhibited, myocardial fibrosis is inhibited, and ventricular remodeling after myocardial infarction is treated, so that cardiovascular diseases, particularly myocardial infarction, are effectively treated. Comprehensive description, RAB31 opens up new directions and new theoretical basis for researching new targets or new treatment strategies of cardiovascular diseases, in particular to preventing or treating myocardial infarction.

Drawings

Fig. 1A shows one of the effects of the knockdown RAB31 on cardiac function of a myocardial infarction mouse in example 2 of the present invention.

FIG. 1B is a graph showing the effect of the knockdown RAB31 on cardiac function of a mouse with myocardial infarction in example 2 of the present invention.

FIG. 1C is a graph showing the effect of the knockdown RAB31 on cardiac function of a myocardial infarction mouse in example 2 of the present invention.

Fig. 2A shows HE staining patterns of the hearts of each group of mice after the RAB31 was knocked out in example 2 of the present invention.

Fig. 2B shows a statistical plot of cardiac index for each group of mice after the RAB31 was knocked out in example 2 of the present invention.

Fig. 3A shows a section of heart tissue from each group of mice examined by Masson staining after the RAB31 was knocked out in example 2 of the present invention.

Fig. 3B shows a statistical plot of heart tissue fibrosis in mice after RAB31 is knocked out in example 2 of the present invention.

FIG. 4 shows the strategy for constructing targeting vectors in example 1 of the present invention. Wherein WILDTYPE ALLET is an allele of RAB31, TARGETING VECTOR is a targeting vector, TARGETED ALLELE is a targeting allele, conditional KO allele is a designated knockout allele (after Neo deletion), constitutive KO allele is a designated knockout allele (after Cre recombination), SDA (self deletion anchor) site, homology arm is a Homology arm, exon of mouse Rab31 is mouse Rab31 exon, and cKO is a knockout region.

FIG. 5 is a schematic representation of the linearized targeting vector of example 1 of the present invention.

Detailed Description

The invention occasionally discovers that by taking a RAB31 gene knockout mouse (Rab 31 ^-/-) as a subject and researching a myocardial infarction model caused by blocking anterior descending branches of left coronary arteries of a mouse heart, the result shows that compared with a WT mouse (Rab 31 ^+/+), the heart ejection fraction, the short-axis shrinkage rate, inflammatory cells, the heart index and the degree of fibrosis of the RAB31 gene knockout mouse are obviously inhibited, the heart function is obviously improved, and the phenotype of the WT mouse is opposite to that of the RAB31 gene knockout mouse. The RAB31 gene can promote and add the occurrence and development of cardiovascular diseases, the RAB31 can be used as a drug target to screen drugs for preventing or treating the cardiovascular diseases, and the inhibitors of the RAB31 can be used for preparing drugs for preventing or treating the cardiovascular diseases. The present invention has been completed on the basis of this finding.

The first aspect of the invention protects the use of a RAB31 inhibitor in the manufacture of a product for the prevention and/or treatment of cardiovascular diseases.

The RAB31 gene is numbered NM-133685 in the NCBI database, ENSMUSG00000056515 in the Ensembl database, located on mouse chromosome 7, 7 exons were identified in total, starting from the start codon ATG of exon 1 to the end of the stop codon TGA of exon 7 (Transcript: ENSMUST 00000070673).

Preferably, the products include pharmaceuticals, nutraceuticals and foods.

Preferably, the RAB31 inhibitor is used alone or in combination with other drugs. I.e. the RAB31 inhibitor may be the only active ingredient or one of the active ingredients of the product. The form of the product is not particularly limited, and can be a common solid, liquid, gel, semifluid, aerosol and the like.

Preferably, the RAB31 inhibitor generally comprises a substance capable of inhibiting transcription or translation of the RAB31 gene, or capable of inhibiting expression or activity of the RAB31 protein. For example, RAB31 may be partially inhibited, i.e., reducing the expression and/or function of RAB31, or may be fully inhibited, i.e., substantially fully eliminating the expression and/or function of RAB 31. For example, RAB31 inhibitors can be nucleic acid molecules, small molecule chemicals, antibody drugs, polypeptides, proteins, nucleic acid constructs, interfering lentiviruses, interfering adeno-associated viruses, and gene editing systems. The nucleic acid molecule can be, but is not limited to, antisense oligonucleotide, double-stranded RNA or shRNA, further can be injected into a human body to silence RAB31 genes by an RNA interference method after being synthesized by a chemical method to treat cardiovascular diseases, can also be used for designing and constructing a mutant of RAB31, enters cells after injection, competes for a primary acting substrate of the RAB31, thereby inhibiting the function of the RAB31 and achieving the purpose of treatment, and the small molecule chemical takes the RAB31 as a target point.

Preferably, the RAB31 inhibitor is a nucleic acid molecule, the target sequence of which comprises the sequence shown in SEQ ID No. 1. More preferably, the nucleic acid molecule comprises the element LoxP-target sequence-LoxP.

GACACGGGGGTTGGGAAATCCAGCATTGTGTGTCGTTTTGTCCAGGATCACTT TGACCACAACATCAGCCCCACTATTGG(SEQ ID NO.1)

More preferably, the nucleic acid molecule further comprises one or more of a selectable marker and SDA. The screening marker is selected from a positive screening marker and a negative screening marker, wherein the positive screening marker is Neo, and the negative screening marker is DTA.

More preferably, the nucleic acid molecule further comprises a first homology arm that is intron No. 1 of the RAB31 gene and a second homology arm that is intron No. 2 of the RAB31 gene.

Further preferred, the nucleic acid molecule comprises the element DTA-LoxP-target sequence-Neo-LoxP, in particular the nucleic acid molecule comprises the element DTA-first homology arm-LoxP-target sequence-SDA-Neo-SDA-LoxP-second homology arm. The LoxP and SDA are conventional sequences of targeting technology, the first homology arm is the intron No.1 of RAB31, and the second homology arm is the intron No. 2 of RAB 31. The size of the insertion 5'-loxp site of the No.1 intron is 50418bp, and the size of the insertion 3' -loxp site of the No. 2 intron is 4444bp.

The Loxp sequence is as follows:

ATAAC TTCGTA TAGCAT ACATTA TACGAA GTTAT(SEQ ID NO.2)

The SDA sequence is as follows:

TAA CTTTAA ATAATG CCAATT ATTTAAAGTTA(SEQ ID NO.3)

first homology arm

TGTTTCT TCATTC TTCGCT CGCTCT AGCCAT AAGCAT CTCCGAAACCCT GCTTTC TCTGTACTGACC GTAGGC TGGACC CCACCG GTATCG GTTATCAGCTAC CTCCCT GGTTTG CAAACT CCATTA GAGGGA GGTGGT AGGCTA CCTTTC CTGGCC TGCAGGGTTCCC AGACAC AGTAGG GAGGGT CTGAAC ACTCAG AGGTGC CCACCCACGTCCAACCCC AGCACG CAGGGC TCTGAA GCAGAT GCTCCA CCTGGC TCTCCTCCCTCA CATGTTTTTAGG TCACAC CCAGGA TCATCC CTATTC TTTACC CCAAAAGTGCTG CTTGGA GAGAGCTGCCCT TAGTTC TCTTGG CTGGAG AACTAT GTACTTCCTTGT ACTTCT TTATTC TTTGGACTCTCT GCCGGG CTCTCA GGGTGT TGTGGTGGCTCC GCCCCT GCCATG CACCAG CCATGACAGATG TTACAT CCTCTC AATTGACATGCT CACCTG GGGCGC ATGCAT GGGGGC GTGGCGGAGAGC TCCACC CTAAGTCCATTC CACAGA TCAGCT GAGCAG AGCCGG CCTGAA GGCCAGCCCTTG AAATACCCTTCA TGGTGA AAGCGC TAAGCT GTTTTA CGATTA TTAACT CCAGTCCTTGCCTTCTGC AGATGG CTCAAG TGAAAC GCCAGC TTTTTT GGTGAG CTAGAGAAGCAAGATGAT CATCTG TCAGAG GGCAAC TTGGGG GAGTTC TCTTCT ATCAAGAGTGTC CTGGGGATTGAA CTCAGT TCATCC ATCATT CTTCAT GTACCC GTTTTCTAAGCC AGTTTC TCAGCCACTTGA TTAGAA TCTTTC AAAAAA AAATTG TATGGATATTTT GTCCAA ATGTAT TTTCCGTGCATC ATGTTT ACGCAA TTGCCT GTAGAGACCAGA AGAGGA TATTAA GTCCCA GATGTCCAGTGG AACTGG GATCAC AGATGTTTGTGG GCTGTT GCGTTG TGGTGC TTGGAA CTGAACCTGGGT CTTCTG CAAGAGCAGCAA GCATTC TTAACT ACTGAG CCATCT TGTCAG CCTAAAACTTTG TTTTTGTTTTTT TGTTTT TGTTTT TAATCA CACCTT TAGGGG TTGGGA ATGTAACTCAGTTGGTAG TAGAGT GGGTGA AGTGCA TGAAGC TCTCTC TCTGGG TTTGATCCCTAGTGATAT GTAAGC CAGGCT TGGTGG TACATC ACTGCA GTCCTG GCACTTGAGATG TGGAGGTATGAT CAGTTC GAGATT ATCCTA ATCCAT AGGAAG CTTGAGGCTAGC TTGCGC TAGGGATACTCT CATAAT AAATAG ATAAAT AAATAA AGTCACATAGGA GAACCA TACACA ACTCTTAAGTAT CTGGGT CACTCA GCTTTT TGTTTCTATGGA AACCTC CCTGGG ACAAAG CAGGAGTGGTTT ATTTTG TCTCAT AGTTTCAAAGGG TCAGTC CATCGT TGGCCA AAAGCT CTGGGGTTTTTA GCTTGA GCACACAATGCA ATTTAA TTGGGT ATCTCT GCCTGT GGGTTC CCCACCCACCTG CCCAGAGGGGGC TCCAGC TATTTC TGCCTA GGCTTA CATTGT TTCTTT CTTCCTCCGTGTGCCTTA TGCTGA TTTGTG AGTCTC AGGCAT GGATTG(SEQ ID NO.4)

Second homology arm

TGTTCG GACAGA ATACCT TGGCAG CAGGAG CATATA GTGGAA GTGAGCTGTTGC TAGCCT TGTAGG GGTCAA GAAACA GAAAAG GAGAAG ACAGGA AGAGGCTGGGAC AAGATA GAGCAT CTAAGG ACACGCCCATTG TGACTC GCTTCC TCTACCTAGACC CTCTTC CTACTT TTCAAT AATGTC CTCATA TGATAA ATGCTT CAAAGGATTCAT CTATTC ATCATC AGGTCA GAGCCC TCATGA TCTAACAGTTTC TGGAAATGCCCC TGTAGA CACACC CACAGG TGTGTT CCACTA TCCCAT TAGGTATCTGTCCACCAC AGCCAA ATTGGA GAATTG AGAGAT CCGTGG ATGTGG AGCACAGAGGGTGTGGCC ACCAGC CAGGTA CATGCT ATGGGG TCACGG TCTCAA GCTTCA

GGATAT CTGAAGACTTAT TATTAT TGTTAT TTCGGG AAGCTC TAGGGG TGCAGC

TTTCAT GCCCGG ACAGATAAGGCC CACCTT ATTTGC CTTGCT GGATTG TCTTAC

CTGCCT GACCCT GTGGAC ATCCCAGTTAAG TCCCTG ACCTAA GAGTGA ATTGTG

ATGGTT TCCTCA GGTCAG AGCACA CAGGTAGCCACA GAGCTG CAAACT TGACTC

AGATCA AACCTA GCAGAG TTGGGT GTATGG CCTTAGTCATAC AGTGCT TGTACA

GCATGG ATGTGC GCTGCC AAAACC AACCAG ACAGGC AGATGACAAAGC AAGCCA

GTCTGT GCTATA AGCTAC CCAGTT CTTCCT ATCAGA ATCATG GTTGTAAAAGAG

CCTCAT TTCTCA TCAGCG GCATTA ACCCAC ATCACC GCAGTG ACACAC

TGATTGTGAAGT GCACTG ACTCAG AGGCAG CTATGC CACGGG CTATGT CCAGGC

ATGAAA GCTGCATCCCTT GAGCTT CCTGCA CAGCCT GCACAC ACGATC ATGTAT

GCAGCA GCTCTG TGAGTAGGTGTT TCTTCC CCACAG AAGTTG TGATCT TGTGAA

ACTTGT TTTACT ATATTA CAGAAGAGTATT TACTGG GGGAGG GTGATG CTTGCA

CACACA GCACAG TGCACA CGTGGA GGTCAGAGGACA ACTTGA AGGAGT TGATTC

TCTCCT TCTATC CTGTGG GTCCTG AGGGTC AAACTCAGGGTT TCAGGC TTGGTG

GCACAC ACTGTG TTATGA GTCTAC TCAGTG TTCCAT GGTGGATGCTAT GTTCTT

GTATAA CTTTCT ATAGAC CTAATT TTTTTT CTTTTC TGTTTT ATTTTTTAAAGA

TTTATT TATTTA TTTCAT GTATGT GAGTAC ACTGTT GCTATC TTCAGA

CACACCAGAAGA GGGCAT TGGATC CCCATT ACAGAT GGTTGT GAGCCA CCATGT

GGTTGC TGGGAATTGAAC TCAGGA CCTCTG GAAGAG CAGTCA GTGTTC TTAACC

ACTGAG CCATCT CTCCAGCCCACT GTTTTT TGGTTG TTTTTG TTGTTG TTGTTT

TTAAAT GAATGA GATACT TTAGCCCATAGA AGAAAG TCTATT AAATTA TTAAAT

ATTCTG TGTATT TGGTTT TTACCC CTTTATGAGCAC TAAGAA AATAAA TGCTTA

TTCAGA GAAAAT AGGTAG ATAAGG ATAGCC AGTAGTTTCTGA CTCATG CCTTGA

AGTCCC TCCTTT GTGATA GAAGCA TACGTG TGTTTT GTTTTTTATCCC TCTGCT

GGATAT TCTGTG TCAACT TGACAC AGGCTA AAGTCA TCTCAG GAGAGAGAACCT

CGATTG TGAACA TTGCTC CATAAA ATAGGA CTATAG GCAAGA CTGTTG

GACATTTTCTTA ATTAGT GATGGA TGTGGG AGGGCC CAGACC ATTGTG GGTGGT

GCGATC TCTGTCCTGGGT TCTATA AGAAAG CAGGCT GAGCAA GCTATG GGGAGC

AAGCCA GTAAGC AGCACCCCTTCA TGGCCT CTGCAT CAGCTT CTGCCT CCAAGT

TCCTGT CCAGTT TGACTT CCTGTCCAGACT TCCTCC AATGCT GAACAG CGATGT

GGAAAT GTAAGC CAGTAA ACCCTT GCTTTGGTTGGT ATTTCA TCATAG CAATGC

AAACCT TGACTT AGGCAA TCCCTA CATTGA TGCAGATAGGTT GAGATT TAGATC

TATTCT TCCATT ACTTGC TGCTGG TATTGA ATGTCA GCACAGGGACTA TGGTAA

GACAGC TTTCTT TTCATA CTGTTT TGAAGT CAAAGT CTTAAA GTGATAAGACATTTTTTT CCTCAT ACTTGT TTTAAT AGTAAG GCTTTA ATATCT AAGGCTAGACATTCAACA TTTCCC TGATAC CCAGAG ACTATT ACTCTA GGACAA TTGACTCTGTTT CTACATGCATTT TCTTTT ATTATG TTTATT TATTTT ATGGGC ATGAGTATTTTG TTTGCA TGTACTTATGCA CAATAC GTAAGT GCAGTG CCTAAG GAAGCCAGAAGA AGGTAT TGGATC CCTTGGAAATGA AGTTAC AGATGG CTGTGA GCTCCTGTGTGA ATGCTG GGGCCC AAACTA AGGTCCTCTGCA AGTGCC TTCGGT ACTCTTAACCTT TGAGCC ATCCCT CCAGTC TACACA CTTTCATGAAGC TTCCAC TACCCCACCTTA GCTCCA GGAACT AGCAGC ACTACC AGCCCT GACCCAGCTTCC TGTGAATCCATG GATAAA AGACAC ACAGAC ACACAC AGGTTG CTCATT TTCAACTAGCCTTCTGAC ACAATT GCTGGG TGCTGC TATCTC CCGTCT GGAAAA GTGTGCCCTTATCAGTAT TCTTAG CTCTGT ACCTCC CACCTG CCCTAA ACTTTA GTTGATCAGTTA CATCTAGTCCCT GTTAAC ATCCCT GACAGA CACACA GGCTGC CACTCCTCCCTC GATCTC ACATGGCTTCTT GGCTTT TCTCTC TCTGAA GCATGG TGAACTCCTCCCTT(SEQ ID NO.5)

Preferably, the RAB31 inhibitor is a nucleic acid construct comprising a nucleic acid molecule as described above.

More preferably, the nucleic acid construct is constructed by PCR with BAC clone RP24-36513 as a template to generate homology arms and knockout regions. Two identical loxP sites are connected to two sides of exon 2 of RAB31 gene and connected to left homology arm, neo screening mark is connected between two self-missing anchors (SDA), 5 ^' end is connected to right loxP site and 3 end ^' end is connected to right homology arm. The sequence of the nucleic acid construct is shown in SEQ ID No.6.

The PCR primer sequences are adopted as follows:

F1:5’-TCCTTATATCAGCGAAACAGCCA-3’(SEQ ID No.7)

R1:5’-AGCAAGCTCTCCCAGACAGTC-3’(SEQ ID No.8)

Preferably, the RAB31 inhibitor is a gene editing system selected from one or more of a Cre-Loxp system, a Flp-FRT system, a Dre-ROX system, a vCre-vloxp system, a sCre-sloxp system, a CRISPR/Cas9 system, a CRISPR/Cas12 system, and/or a CRISPR/Cas-derived single base editing system. The gene editing system uses the sequence shown in SEQ ID No.1 as a target sequence (a knockout region or cKO region) for knockout. In certain embodiments, the RAB31 inhibitor is a Cre-Loxp system comprising a targeting vector as described above that knocks out the RAB31 gene under the action of Cre recombinase.

Preferably, the RAB31 inhibitor has the effect of preventing and/or treating myocardial infarction by at least one of:

Reducing inflammatory infiltration of myocardial cells;

reducing cardiomyocyte depletion;

Inhibiting cardiac hypofunction;

inhibiting myocardial fibrosis;

improving heart reconstruction after myocardial infarction.

In the acute phase of myocardial infarction (AMI), the myocardium in the infarct zone is necrotizing coagulum, and the myocardium is engorged with congestion and edema, accompanied by infiltration of inflammatory cells. After the acute phase, as collagen matrix supporting cardiomyocytes is decomposed, necrotic cardiomyocytes slip off, necrotic tissue stretches and becomes thin later, which is one of the causes of early left ventricular dilatation, whereas non-necrotic area myocardial interstitium is also reconstructed due to the influence of neuroendocrine, resulting in impaired systolic and diastolic functions of the heart and ultimately heart failure. Only a small amount of myocardial cells divide and proliferate after myocardial infarction, and myocardial tissues cannot be effectively and completely repaired, so that myocardial in an infarcted area can only proliferate through fibrous tissues, ventricular remodeling is replaced by scar tissues without contraction function, and serious arrhythmia, cardiac insufficiency and even death are caused. The application discovers that the RAB31 gene can be knocked out to obviously inhibit Left Ventricular Ejection Fraction (LVEF) and left ventricular short axis contraction rate (LVFS), which indicate that heart function decline caused by myocardial infarction is obviously inhibited after the RAB31 gene is knocked out, and through HE staining, the RAB31 gene can be knocked out to reduce myocardial cell number reduction, reduce inflammatory infiltration and reduce heart index, and through Masson staining, the RAB31 gene can be knocked out to inhibit myocardial fibrosis, thereby comprehensively realizing improvement and treatment of ventricular remodeling after myocardial infarction, and effectively treating cardiovascular diseases, especially myocardial infarction.

In another aspect the invention also provides a nucleic acid molecule for reducing the expression of the RAB31 gene, the target sequence of which comprises the sequence shown in SEQ ID NO. 1.

Preferably, loxp is linked upstream of the target sequence and loxp is linked downstream of the target sequence to form loxp-target sequence-loxp.

More preferably, a first homology arm is connected to the upstream of the first Loxp segment, a second homology arm is connected to the downstream of the second Loxp segment, and a positive screening marker is arranged between the two Loxp sequences. The positive selection marker is Neo. The positive selection marker is flanked by SDA sites.

Further preferably, a negative selection marker is attached upstream of the first homology arm. The negative selection marker is DTA.

In a specific embodiment, the nucleic acid molecule comprises a DTA-first homology arm-LoxP-target sequence-SDA-Neo-SDA-LoxP-second homology arm.

In a further aspect the invention also protects a substance associated with a nucleic acid molecule as described above, said substance comprising one or more of 1) a nucleic acid construct comprising a nucleic acid molecule as described above, 2) a gene editing system targeting a nucleic acid molecule as described above and knocking out, or 1) a nucleic acid construct, 3) a cell line comprising a nucleic acid molecule as described above, or 1) a nucleic acid construct, or 2) a nucleic acid construct obtained by transfection of a host cell with the gene editing system, 4) a pharmaceutical composition comprising a nucleic acid construct as described in 1), or 2) a gene editing system as described in 3) a cell line as described in 3). Preferably, the nucleic acid construct (targeting vector) may be obtained by cloning the nucleic acid molecule encoding the above into a known vector. The known vector is bacterial artificial chromosome BAC. Those skilled in the art can construct the vector by using a conventional targeting expression vector method. The nucleic acid construct is formed by inserting the target sequence into a plasmid containing Loxp, neo and DTA by homologous recombination. The nucleic acid construct comprises a DTA-first homology arm-LoxP-target sequence-SDA-Neo-SDA-LoxP-second homology arm, and the sequence of the formed nucleic acid construct is shown as SEQ ID NO.6.

Preferably, the gene editing system is a Cre-Loxp system comprising the nucleic acid construct (targeting vector) described above.

Preferably, the cell line refers to the use of ES cells as host cells, and the RAB31 gene in the host cells is knocked out by transfecting the gene editing system. The ES cells are C57BL/6NES cells. The targeting vector in the gene editing system is linearized, as by digestion, and the host cells are transfected by electroporation.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. By pharmaceutically acceptable adjuvant is meant that when the drug is properly administered to an animal or human, they do not produce adverse, allergic or other untoward reactions. The pharmaceutically acceptable excipients should be compatible with the RAB31 inhibiting substance, i.e. be able to be blended therewith without substantially reducing the effect of the RAB31 inhibiting substance in the usual case. The pharmaceutically acceptable auxiliary materials are selected from one or more of carriers, diluents, binders, lubricants and wetting agents. Specific examples of some substances which can be pharmaceutically acceptable carriers, diluents, binders, lubricants and wetting agents are sugars such as lactose, dextrose and sucrose, starches such as corn starch and potato starch, celluloses and derivatives thereof such as sodium methyl cellulose, ethyl cellulose and methyl cellulose, tragacanth powder, malt, gelatin, talc, solid lubricants such as stearic acid and magnesium stearate, calcium sulfate, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter, polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol, alginic acid, emulsifying agents such as Tween, wetting agents such as sodium lauryl sulfate, colorants, flavoring agents, compressed tablets, stabilizers, antioxidants, preservatives, pyrogen-free water, isotonic saline solution, phosphate buffer and the like. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration. The formulation of the composition is one or more of solution, injection, spray, nose drops, aerosol, powder fog, tablet, capsule and granule. The composition can be introduced into the body by injection, nasal drip, eye drip, permeation, absorption, physical or chemical mediated methods, such as intramuscular, intradermal, subcutaneous, intravenous, mucosal tissue, or mixed or encapsulated with other substances. Preferably, the administration is via the abdominal cavity. The medicament may also be used in combination with other therapeutic means including surgery, radiation therapy, chemotherapy, targeted therapy.

In a further aspect the invention also provides the use of a nucleic acid molecule as described above or a substance as described above for the preparation of a kit for preventing or treating cardiovascular disease and/or for reducing the expression of the RAB31 gene.

Preferably, the product for preventing or treating cardiovascular diseases has at least one of the following effects of reducing inflammatory infiltration of myocardial cells, reducing myocardial cell loss, inhibiting cardiac hypofunction, inhibiting myocardial fibrosis and improving ventricular remodeling after myocardial infarction.

In another aspect, the invention also provides a method for screening medicaments for preventing and/or treating cardiovascular diseases and myocardial infarction, which comprises taking RAB31 as a medicament target, and searching for substances capable of inhibiting or blocking the expression and/or the function of the RAB31 as candidate medicaments.

Preferably, an in vitro cell model or an animal model of the overexpression of the RAB31 gene is constructed, the in vitro cell model or the animal model is applied with a drug to be selected, and then the content of the RAB31 is detected. The cells may be from a mammal

The test person can determine whether the drug to be selected is a therapeutically significant drug by detecting the content of RAB 31. Generally, the content of RAB31 can be reduced by 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% compared to the control group, respectively, and can be judged as a therapeutically significant drug.

In the present invention, the cardiovascular disease is ischemic heart disease, including myocardial infarction, arrhythmia, coronary atherosclerotic heart disease. Preferably, myocardial infarction. Myocardial infarction (myocardial infraction, MI) refers to a blockage of the coronary arteries, interruption of blood flow, and local myocardial necrosis of the heart in its blood supply area due to persistent and severe ischemia.

Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.

Before further describing embodiments of the invention, it is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is intended to be in the nature of specific embodiments and is not intended to be limiting of the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

In the following examples of the application, C57BL/6 mice male mice were SPF grade, weighing 20-25+ -3 g. Provided by Shanghai Laike laboratory animal center, laboratory animal use license number SYXK (Min) 2014-0001.

EXAMPLE 1 construction of RAB31 knockout mice

The construction method of the RAB31 knockout targeting vector in the invention is shown in FIG. 4, and a targeting structure crossing an exon of RAB31 is utilized to generate a RAB31 flox mouse. Briefly, this structure includes a loxP-target sequence-loxP cassette to construct a targeting vector for RAB 31. Electroporation of the constructed targeting vector into embryonic stem cells to form ES clones, and PCR screening of the ES clones. The correct clone was obtained, the clone was injected into blastula and finally homozygous mice were constructed to obtain the RAB31 knockout.

1.1 Construction of Gene knockout targeting vector

The mouse RAB31 gene is located on mouse chromosome 7. 7 exons were identified altogether, starting from the start codon ATG of exon 1 to the end of the stop codon TGA of exon 7 (Transcript: ENSMUST 00000070673).

Exon 2 was selected as a conditional knockout region (cKO region) whose deletion should lead to loss of function of the mouse RAB31 gene.

The sequence of exon 2 is shown in SEQ ID No. 1.

The homology arm and conditional knockout region (cKO region) of the RAB31 gene was generated by PCR using RP24-36513 from the C57BL/6N mouse library BAC clone as template, using the PCR primer sequences:

F1:5’-TCCTTATATCAGCGAAACAGCCA-3’(SEQ ID No.7)

R1:5’-AGCAAGCTCTCCCAGACAGTC-3’(SEQ ID No.8)

the nucleic acid molecule produced by PCR comprises the elements of a first homology arm-exon 2 and a second homology arm.

The construction process of the targeting vector comprises inserting two LoxP loci on both sides of exon 2 of the mouse RAB31 gene, inserting two SDA (self-deletion anchor) loci and Neo positive selection marker elements, and finally inserting DTA negative selection marker elements at 5 ^' ends of 5 ^' homology arms (namely first homology arms). The targeting vector comprises the following elements of DTA-first homology arm-LoxP-exon 2-SDA-Neo-SDA-Loxp-second homology arm. Neo is a neomycin resistance gene as a positive selection marker, DTA is a toxin gene as a negative selection marker, and SDA is an self-deleted anchor point.

LoxP sequence is shown as SEQ ID NO.2, SDA sequence is shown as SEQ ID NO.3, the first homology arm is shown as SEQ ID NO.4, the second homology arm is shown as SEQ ID NO.5, and the sequence of the targeting vector is shown as SEQ ID NO. 6.

Linearizing the targeting vector by adopting enzyme, wherein a schematic diagram of the linearized targeting vector is shown in fig. 5.

1.2, ES targeting and screening, namely introducing the linearized targeting vector into the ES cells of a mouse by an electrotransformation method, culturing overnight, and then carrying out positive and negative screening, wherein positive recombinant ES cells are reserved for the intracavitary injection of a recipient mouse.

1.3, Blastula injection, the screened positive cell clone with homologous recombination is injected into blastula of C57BL/6N donor mice for 3.5 days, and the blastula is transplanted into uterus of CD-1 pseudopregnant mice to produce F0 generation positive chimeric mice.

1.4 Obtaining homozygotes F0-positive mice were bred with tissue-specific Cre mice (WT wild type mice with Cre enzyme, sai Biotech Co., ltd.) at 1% with 2% Cre to obtain F1-mice (flox ^/+, cre) and F1-mice (flox ^/+), and F1-mice (flox ^/+, cre) and F1-mice (flox ^/+)) were identified and bred at 1% with 2% Cre to obtain F2-mice (flox ^/flox, cre) and F2-mice (flox ^/flox), F2-mice (flox ^/flox) namely Rab31 ^-/- (KO) mice, which were bred to 8-10 weeks old for the next experiment.

Flox ^/+ indicates that the locus is heterozygous, i.e., one allele carries a flox site and the other is a wild-type allele, flox ^/flox indicates that the locus is double flox and both alleles carry a flox site.

Example 2

In this example, mice were examined for cardiac function, cardiac pathology and myocardial fiber changes following RAB31 gene knockout.

Comprises the following steps:

2.1 Experimental materials

2.1.1 Laboratory animals

RAB31 knockout mice constructed in example 1 were raised and bred in the SPF class animal center of my school.

Animal experiments were performed according to the "guidelines for treatment of laboratory animals" issued by the national science and technology department in 2006 and the "guidelines for care and use of laboratory animals" issued by the national institutes of health in the united states.

Mice were placed in specific nonpathogenic laboratory animal centers at the university of Fujian, raised in SPF-class laboratories, fed with free movement, drinking water, indoor ventilation light was sufficient, room temperature was controlled at (25+ -1) °C, relative humidity was around 60%, 12h light/dark cycles, and mice were allowed to adapt to these conditions for at least 7 days prior to the experiment. Keeping the raising environment quiet without disturbance.

2.1.2 Experimental drugs and Main reagents

Eosin staining solution (Cat No. G1100-500 of Beijing Soy Bao technology Co., ltd.), hematoxylin staining solution (Cat No. G1140-500 of Beijing Soy Bao technology Co., ltd.), isoflurane (Cat No. R510-22 of Ruiword life technology Co., shenzhen Co., ltd.), paraformaldehyde (Cat No. LA0427 of Beijing Soy Bao technology Co., ltd.), absolute ethyl alcohol (Cat No.64-17-5 of West Long chemical Co., ltd.), xylene (Cat No.2052-46-2 of national pharmaceutical composition chemical Co., ltd.).

2.1.3 Main experimental instrument

The device comprises a small animal ultrasonic imaging system Vevo2100 (Fuji film investment Co., ltd.), an inhalation type small animal anesthesia machine (Ruiword life technology Co., ltd. In Shenzhen, germany), a pathological microtome (Leca Co., germany), a paraffin embedding machine (Hubei Xiaoshi medical electronic technology Co., ltd.), and an automatic biological tissue dehydrator (Hubei Xiaoshi medical electronic technology Co., ltd.).

2.2 Experimental methods

2.2.1 Grouping of animals and intervention

Male wild-type WT mice (Rab 31 ^+/+) of 8-10 weeks of age were randomly divided into 2 groups, wt+sham group (n=6), wt+MI group (n=6). Wherein the WT+MI group means that the WT mice had Myocardial Infarction (MI) model by ligating the left anterior descending branch (LAD) of the heart, i.e., MI surgery+ and Rab31 ^+/+, and the WT+Sram means that the WT mice did not ligate the LAD after chest opening, i.e., MI surgery-and Rab31 ^+/+.

Meanwhile, male KO mice (Rab 31 ^-/-) obtained in example 1, which were 8-10 weeks old, were randomly divided into 2 groups, ko+sham group (n=6), ko+mi group (n=6). Wherein, KO+MI group means that KO mice cause Myocardial Infarction (MI) model by ligating anterior descending branch (LAD) of left coronary artery of heart, namely MI surgery+ and Rab31 ^-/-, KO+sham means that KO mice do not ligate LAD after opening chest, namely MI surgery-and Rab31 ^-/-.

Ligating the anterior descending branch of the left coronary artery to establish a Myocardial Infarction (MI) model, namely, a mouse is in a supine position, muscles are passively separated between the third rib and the fourth rib on the left side, a hemostatic forceps is used for expanding the third rib and the fourth rib, after the heart is rapidly extruded, a 6-0 band suture needle ligates the anterior descending branch of the left coronary artery, the ligature position is 1mm below the left auricle, the needle insertion depth is 0.5mm, the heart muscle in a myocardial infarction area below the ligature part is changed into pale color in the operation, whether ligature is successful or not is judged, and whether the modeling is successful or not is further judged by connecting an electrocardiogram. The intraperitoneal injection of penicillin is routinely administered after operation, 10 ten thousand per U per day, for 3 consecutive days. And disinfect the incision skin to prevent infection.

Each group of cardiac functions was measured at week 2 post-surgery and the experiment was terminated after 2 weeks.

2.2.3 Ultrasonic detection of cardiac function in mice

Mice were examined for cardiac function in the second week post-operative (day 7 post-myocardial infarction) using a Vevo 2100 animal sonicator. The experimental animal is fixed on a 37 ℃ constant temperature heating plate in a supine position under inhalation anesthesia of 2% isoflurane, and after chest dehairing, the probe is placed on the chest, and the probe frequency is 400MHz. Collecting a parasternal long-axis tangential plane and a left-room short-axis tangential plane, and recording the left-room movement condition by applying M-type ultrasonic to the rear edge of papillary muscles. The measurement indexes include the inner diameter of the end diastole (LVED; d) and the inner diameter of the end systole (LVES; d) of the left ventricle, and the Left Ventricular Ejection Fraction (LVEF) and the short axis systole (LVFS) of the left ventricle and the like are calculated to compare the heart function changes of the mice in each group.

Left Ventricular Ejection Fraction (LVEF) and short axis shrinkage (LVFS) are calculated as follows:

LVEF(%)=(LV Vol;d–LV Vol;s)/LV Vol;d×100%

LVFS(%)=(LVID;d-LVID;s)/LVID;d×100%

2.2.4 HE staining

Taking a mouse heart on day 14 after myocardial infarction operation, fixing the heart in 4% paraformaldehyde for 24 hours, then adopting absolute ethyl alcohol with low concentration to high concentration to carry out tissue dehydration treatment, then putting a tissue block into dimethylbenzene for transparency, replacing medium alcohol of the tissue block with dimethylbenzene, carrying out paraffin embedding treatment on vascular tissues after waxing, and cooling and solidifying the vascular tissues into blocks. The embedded wax block is fixed on a slicing machine, cut into 4 mu m slices, the cut slices are put into 37 ℃ water for ironing, then are stuck on a glass slide, and are baked for 1h at 60 ℃ to prepare paraffin sections. Tissue sections were transparently processed in xylene I, II and rehydrated in ethanol gradient. The samples were stained in hematoxylin solution for 1min, stained in eosin solution for 10s, air-dried, gel-sealed with neutral resin, and observed under a microscope for pathological changes of the heart.

2.2.5 Masson staining

Taking paraffin sections of mice heart fixed 14 days after myocardial infarction operation, carrying out tissue dehydration, embedding and slicing, carrying out xylene transparent treatment on the sections, carrying out gradient dewaxing on ethanol, and washing with tap water and distilled water in sequence. Then using Regaud hematoxylin dye liquor or Weigert hematoxylin semen to dye the nucleus for 5-10min, fully washing with water, using Masson ponceau acid reddish solution for 5-10min, using 2% glacial acetic acid aqueous solution to soak and wash for a moment, using 1% phosphomolybdic acid aqueous solution to differentiate for 3-5min, directly using aniline blue or light green liquor to dye for 5min without washing, then using 0.2% glacial acetic acid aqueous solution to soak and wash, using 95% alcohol and absolute alcohol to dehydrate, using xylene to make transparent and neutral gum seal, and observing the change of fibrosis degree under a lens.

2.2.6 Statistical analysis

Experimental data are expressed as mean ± standard deviation (x±s), analyzed by SPSS18 software, t-tests were used for comparison between the two sets of averages, single-factor analysis of variance was used for comparison between the sets of averages, and P <0.05 was considered statistically significant.

2.3 Experimental results

2.3.1 Effect of knockout of RAB31 Gene on cardiac function in myocardial infarction mice

The heart function of the mice was examined the second week after surgery and RAB31 knockdown was performed using a small animal ultrasound to measure the end-diastole inner diameter of the left ventricle (LVED; d), the end-systole inner diameter of the left ventricle (LVES; d), the Left Ventricular Ejection Fraction (LVEF), and the left ventricular short axis systole (LVFS).

Fig. 1A is a cardiac ultrasound chart, fig. 1B is a statistical chart of Left Ventricular Ejection Fraction (LVEF), and fig. 1C is a statistical chart of table left ventricular short axis shrinkage (LVFS). LVEF and LVFS can reflect heart function, and low values indicate poor heart function.

From FIGS. 1A-1C, it is evident that both LVEF and LVFS were significantly reduced in the WT+MI group compared to the WT+Sram group, the difference was statistically significant (P < 0.05), and both LVEF and LVFS were significantly increased in the KO+MI group compared to the WT+MI group, the difference was statistically significant (P < 0.05), suggesting that cardiac hypofunction due to myocardial infarction was significantly inhibited after RAB31 knockout.

2.3.2 Effect of knockout Rab31 on cardiac pathological morphology in mice with myocardial infarction

The heart pathology changes of each group of mice were compared by HE staining and the results are shown in fig. 2A.

As can be seen from FIG. 2A, the WT+MI group showed a large number of myocardial cells missing and inflammatory cells infiltrating as compared with the WT+sham group, while the KO+MI group showed a large number of myocardial cells present and inflammatory infiltration reduced as compared with the WT+MI group.

The heart index change of each group of mice was further measured, i.e. the heart of the mice on day 14 after acute myocardial infarction was taken, excess vascular connective tissue at the bottom of the heart was trimmed in ice PBS, and the water on the heart was dipped with filter paper, weighed and HW was recorded. Tibia was removed, tibia length TL was measured with vernier calipers, and heart index (heart index) =heart weight (HW)/Tibia Length (TL).

The heart will increase in weight after myocardial infarction, and the severity of myocardial infarction can be reflected by calculating the heart index change.

The cardiac index results for the different groups are shown in fig. 2B.

From 2B, the cardiac index was significantly increased in the wt+mi group compared to the wt+sham group. In contrast, the ko+mi group showed a statistically significant (P < 0.05) decrease in cardiac index compared to the wt+mi group, indicating that RAB31 knockout inhibited cardiac index elevation due to myocardial infarction.

Taken together, RAB31 knockdown can ameliorate heart pathological changes caused by myocardial infarction.

2.3.3 Effects of knockout of RAB31 on myocardial fibrosis in mice with myocardial infarction

The Masson staining was performed to examine the change in cardiac fibrosis in each group of mice, the staining results are shown in FIG. 3A, and the statistical results are shown in FIG. 3B.

From FIG. 3A, it can be seen that the deposition of collagen in the myocardial tissue of the WT+MI group was significantly reduced compared to the WT+Shom group, and the deposition of collagen in the KO+MI heart tissue was significantly reduced compared to the WT+MI group, indicating that RAB31 knockout could improve myocardial fibrosis caused by myocardial infarction.

As can be seen from fig. 3B, the areas of the groups were further counted, and it was found that RAB31 knockout could improve myocardial fibrosis caused by myocardial infarction.

Taken together, the invention discovers that the RAB31 knockout can obviously reduce the heart function decline caused by MI, has the effect of relieving the myocardial fibrosis caused by MI, and prompts that the RAB31 is an important target for treating ventricular remodeling after myocardial infarction.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (8)

1. The application of the RAB31 inhibitor in preparing medicines for preventing and/or treating myocardial infarction is characterized in that the RAB31 inhibitor is selected from nucleic acid molecules, the nucleic acid molecules comprise the following elements of DTA-first homology arm-LoxP-exon 2-SDA-Neo-SDA-LoxP-second homology arm, DTA, loxP, SDA is a targeting technology conventional sequence, the first homology arm is an intron 1 of RAB31, the second homology arm is an intron 2 of RAB31, the exon 2 is a sequence shown as SEQ ID NO:1, the first homology arm is a sequence shown as SEQ ID NO:4, and the second homology arm is a sequence shown as SEQ ID NO: 5.
2. Use of a RAB31 inhibitor for the preparation of a medicament for the prevention and/or treatment of myocardial infarction, said RAB31 inhibitor being selected from the group consisting of nucleic acid constructs comprising the nucleic acid molecule as set forth in claim 1.
3. Use of a RAB31 inhibitor for the preparation of a medicament for the prevention and/or treatment of myocardial infarction, said RAB31 inhibitor being selected from the group consisting of a gene editing system comprising the nucleic acid construct of claim 2.
4. Use of a RAB31 inhibitor for the preparation of a medicament for the prevention and/or treatment of myocardial infarction, said RAB31 inhibitor being selected from a cell line comprising the nucleic acid molecule of claim 1 or the nucleic acid construct of claim 2 or the gene editing system of claim 3 transfected into a host cell.
5. Use of a RAB31 inhibitor for the preparation of a medicament for the prevention and/or treatment of myocardial infarction, the RAB31 inhibitor being selected from the group consisting of a pharmaceutical composition comprising the nucleic acid molecule of claim 1 or the nucleic acid construct of claim 2 or the gene editing system of claim 3 or the cell line of claim 4, and a pharmaceutically acceptable adjuvant.
6. 6. The use according to any one of claims 1to 5, wherein the RAB31 inhibitor is effective in preventing and/or treating cardiovascular disease by at least one of:

   Reducing inflammatory infiltration of myocardial cells;

   reducing cardiomyocyte depletion;

   Inhibiting cardiac hypofunction;

   inhibiting myocardial fibrosis;

   improving heart reconstruction after myocardial infarction.
7. 7. A nucleic acid molecule for reducing RAB31 gene expression is characterized by comprising the following elements of DTA-a first homology arm-LoxP-exon 2-SDA-Neo-SDA-LoxP-a second homology arm, DTA, loxP, SDA is a targeting technology conventional sequence, the first homology arm is an intron 1 of RAB31, the second homology arm is an intron 2 of RAB31, the exon 2 is a sequence shown as SEQ ID NO.1, the first homology arm is a sequence shown as SEQ ID NO. 4, and the second homology arm is a sequence shown as SEQ ID NO. 5.
8. 8. Use of a nucleic acid molecule according to claim 7 for the preparation of a kit for reducing RAB31 gene expression.