Use of RAB31 inhibitors in the preparation of products for preventing and / or treating hypertension

Abstract

The present invention discloses the use of RAB31 in preparing a product for preventing and / or treating hypertension. The present invention finds that inhibiting the expression of the RAB31 gene can significantly inhibit the increase in blood pressure of mice after AngII stimulation, inhibit the increase in the thickness of the abdominal aorta media, inhibit the increase in the pulse wave propagation velocity, inhibit cardiac systolic dysfunction, and reduce hypertension, indicating that RAB31 is a new target or new treatment strategy for studying hypertension.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a RAB31 inhibitor in preparing a product for preventing and / or treating hypertension. Background Art

[0002] Hypertension is a major chronic disease, and long-term hypertension increases the risk of cardiovascular and cerebrovascular diseases. According to data released by the WHO in 2021, 1.4 billion people in the world suffer from hypertension, but only 14% of patients have their blood pressure under control. The latest survey data on hypertension in China in 2018 showed that the weighted prevalence of hypertension in adults aged ≥18 years in my country was 27.5%, and the prevalence was generally increasing, and there was a trend of younger age. Currently commonly used drugs include diuretics, beta-blockers, ACE inhibitors, ARBs, calcium channel blockers, and centrally acting drugs. These drugs have different mechanisms of action and can be used alone or in combination. Despite the continuous development of their own prevention and treatment methods and the combined use of different antihypertensive drugs, the awareness rate and treatment rate of hypertension were 51.6% and 45.8%, respectively, and the control rate was only 16.8%, which was still at a low level overall, and the prevention and control situation was severe. At the same time, the above-mentioned drug treatments also have some limitations and shortcomings, such as hypotension, dizziness, headache, constipation, bradycardia, etc. In addition, long-term use of hypertension drugs may also lead to adverse reactions, such as renal damage, metabolic abnormalities, and drug tolerance. The complexity of the pathogenesis of hypertension is the main reason for the existence of drug resistance and adverse reactions in current drug treatments. It is urgent to discover new therapeutic targets and elucidate new pathogenesis.

[0003] The Rab family is a highly conserved class of small GTPases that are responsible for regulating multiple cellular functions in cells, including membrane transport, endoplasmic reticulum-Golgi transport, endocytosis, and vesicle transport. Currently, there are more than 70 known Rab proteins, and Rab family members are distributed in different subcellular regions of cells. For example, Rab5 is involved in the formation of early endosomes, Rab7 is involved in late endosome-lysosome transport, and Rab11 is involved in the transport of circulating endosomes.

[0004] RAB31 is a member of the Rab family and a small GTPase. The protein is highly expressed on the plasma membrane and endoplasmic reticulum membrane and is involved in the regulation of cell division. Current studies have shown that RAB31 plays an important role in regulating cell endocrine pathways, intracellular transport and autophagy.

[0005] RAB31 has been reported to be closely related to the occurrence and development of malignant tumors such as breast cancer and cervical cancer, but there have been no reports on its impact on cardiovascular diseases such as hypertension. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use of a RAB31 inhibitor in the preparation of a product for preventing and / or treating hypertension, in order to solve the above-mentioned problems.

[0007] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0008] The first aspect of the present invention protects the use of the RAB31 gene as a target in screening products for the prevention, treatment or diagnosis of hypertension.

[0009] The second aspect of the present invention protects the use of a RAB31 inhibitor in the preparation of a product for the prevention or treatment of hypertension.

[0010] The third aspect of the present invention protects the use of a nucleic acid molecule or a substance related to a nucleic acid molecule in the preparation of a product for preventing and / or treating hypertension, wherein the target sequence of the nucleic acid molecule comprises the sequence shown in SEQ ID NO.1.

[0011] The fourth aspect of the present invention protects the use of a RAB31 inhibitor in preparing a kit for reducing the expression of the RAB31 gene.

[0012] The fifth aspect of the present invention protects a method for screening drugs for preventing and / or treating hypertension, the method comprising: taking RAB31 as a drug target, searching for a substance that can inhibit or block the expression and / or function of RAB31 as a candidate drug

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention first discovered that RAB31 can be used as a potential target site for screening drugs for preventing or treating hypertension. In addition, it was found that inhibiting the expression of the RAB31 gene can significantly inhibit the increase in blood pressure, inhibit the increase in the thickness of the abdominal aorta media, inhibit the increase in the pulse wave propagation velocity, and inhibit the cardiac contractile dysfunction of mice after AngII stimulation, thereby comprehensively reducing hypertension. Overall, RAB31 has opened up a new direction and new theoretical basis for studying new targets or new treatment strategies for hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is the construction strategy of the targeting vector in Example 1 of the present invention.

[0016] Figure 2 Shown is a schematic diagram of the linearized targeting vector in Example 1 of the present invention.

[0017] Figure 3 The graph shows the systolic blood pressure of each group of mice before surgery and at the 1st, 2nd, 3rd and 4th week after surgery in Example 1 of the present invention.

[0018] Figure 4 The graph shows the diastolic blood pressure results of each group of mice before surgery, and at the 1st, 2nd, 3rd, and 4th week after surgery in Example 1 of the present invention.

[0019] Figure 5 The graph shows the mean arterial pressure results of each group of mice before surgery, and at the 1st, 2nd, 3rd, and 4th week after surgery in Example 1 of the present invention.

[0020] Figure 6 Shown are ultrasound images of abdominal aorta changes in each group of mice in Example 1 of the present invention, as well as statistical graphs of abdominal aorta pulse wave propagation velocity and abdominal aorta media thickness.

[0021] Figure 7 Shown are the HE staining results of the pathological changes in the abdominal aorta of each group of mice in Example 1 of the present invention.

[0022] Figure 8 Shown are ultrasound images of cardiac changes in each group of mice in Example 1 of the present invention and statistical graphs of left ventricular ejection fraction and left ventricular short-axis shortening rate. DETAILED DESCRIPTION

[0023] The present invention accidentally discovered that RAB31 knockout mice (RAB31 KO) were used as the object, and a pre-set osmotic mini-pump containing angiotensin II (AngII) was implanted subcutaneously for research. The results showed that after AngII stimulation, the blood pressure, abdominal aortic media thickness, and pulse wave propagation velocity of WT mice were significantly increased, and the cardiac contraction function was significantly reduced. The phenotype of RAB31 knockout mice was opposite to that of WT mice. Compared with WT, the blood pressure, abdominal aortic media thickness, and pulse wave propagation velocity of RAB31 knockout mice after AngII stimulation were significantly reduced, and the cardiac contraction function of mice was significantly increased. It shows that RAB31 can be used as a drug target to screen drugs for preventing or treating hypertension, and RAB31 inhibitors can be used to prepare drugs for preventing or treating hypertension. The present invention was completed on this basis.

[0024] The first aspect of the present invention protects the use of the RAB31 gene as a target in screening products for the prevention, treatment or diagnosis of hypertension.

[0025] The use of the RAB31 gene in screening for the prevention and treatment of hypertension specifically refers to: using RAB31 as the target of action, screening drugs or preparations to find drugs that can inhibit the expression of RAB31 as alternative drugs for the prevention and treatment of hypertension.

[0026] Preferably, the products include but are not limited to medicines, health products and foods.

[0027] The use of the RAB31 gene as a target for preparing hypertension diagnostic drugs specifically refers to: using the RAB31 gene expression product as a hypertension diagnostic indicator in the preparation of hypertension diagnostic drugs.

[0028] The second aspect of the present invention protects the use of a RAB31 inhibitor in the preparation of a product for the prevention or treatment of hypertension.

[0029] Preferably, the RAB31 inhibitor generally includes a substance that can inhibit the transcription or translation of the RAB31 gene, or can inhibit the expression or activity of the RAB31 protein. For example, RAB31 can be partially inhibited, that is, the expression and / or function of RAB31 is reduced; it can also be completely inhibited, that is, the expression and / or function of RAB31 is substantially completely eliminated. For another example, the RAB31 inhibitor can be a nucleic acid molecule, a small molecule chemical drug, an antibody, a polypeptide, a protein, a nucleic acid construct, an interfering lentivirus, an interfering adeno-associated virus, and an editing system.

[0030] More preferably, the RAB31 inhibitor is a nucleic acid molecule. The nucleic acid molecule can be selected from, but not limited to, antisense oligonucleotides, double-stranded RNA or shRNA. Further, after the double-stranded RNA is synthesized by chemical methods, it is injected into the human body to silence the RAB31 gene by RNA interference to treat hypertension; a mutant of RAB31 can also be designed and constructed, which enters the cell after injection to compete for the substrate of the RAB31 prototype, thereby inhibiting the function of RAB31 and achieving the purpose of treatment. Specifically, the target sequence of the nucleic acid molecule comprises the sequence shown in SEQ ID No.1. The target sequence is exon 2 of RAB31.

[0031] GACACGGGGGTTGGGAAATCCAGCATTGTGTGTCGTTTTGTCCAGGATCACTTTGACCACAACATCAGCCCCACTATTGG(SEQ ID NO.1)

[0032] Further preferably, the nucleic acid molecule comprises the following elements: LoxP-target sequence-LoxP. LoxP is a sequence commonly used for targeting, and the sequence is as follows: Two LoxPs are in the same direction.

[0033] ATAAC TTCGTA TAGCAT ACATTA TACGAA GTTAT(SEQ ID NO.2)

[0034] More preferably, the nucleic acid molecule further comprises one or both of a screening marker and SDA. The screening marker is selected from a positive screening marker and a negative screening marker, the positive screening marker is Neo, and the negative screening marker is DTA. Neo is a neomycin resistance gene, DTA is a toxin gene, and SDA is a self-deletion anchor, generally located on both sides of Neo. Preferably, the nucleic acid molecule comprises the following elements: DTA-LoxP-target sequence-SDA-Neo-SDA-LoxP. The sequence of SDA is as follows.

[0035] TAACTTTAAAATAATG CCAATT ATTTAAAGTTA(SEQ ID NO.3)

[0036] More preferably, the nucleic acid molecule further comprises a first homology arm and a second homology arm, and the sequences of the first homology arm and the second homology arm are as follows. The first homology arm is the first intron of RAB31, and the second homology arm is the second intron of RAB31. The size of the first intron inserted into the 5'-loxp site is 50418 bp, and the size of the second intron inserted into the 3'-loxp site is 4444 bp. The sequences of the first homology arm and the second homology arm are as follows.

[0037] First homology arm

[0038] TGTTTCT TCATTC TTCGCT CGCTCT AGCCAT AAGCAT CTCCGAAACCCT GCTTTCTCTGTACTGACC GTAGGC TGGACC CCACCG GTATCG GTTATCAGCTAC CTCCCT GGTTTG CAAACTCCATTA GAGGGA GGTGGT AGGCTA CCTTTC CTGGCC TGCAGG GTTCCC AGACAC AGTAGG GAGGGTCTGAAC ACTCAG AGGTGC CCACCC ACGTCCAACCCC AGCACG CAGGGC TCTGAA GCAGAT GCTCCACCTGGC TCTCCT CCCTCA CATGTTTTTAGG TCACAC CCAGGA TCATCC CTATTC TTTACC CCAAAAGTGCTG CTTGGA GAGAGCTGCCCT TAGTTC TCTTGG CTGGAG AACTAT GTACTT CCTTGT ACTTCTTTATTC TTTGGACTCTCT GCCGGG CTCTCA GGGTGT TGTGGT GGCTCC GCCCCT GCCATG CACCAGCCATGACAGATG TTACAT CCTCTC AATTGA CATGCT CACCTG GGGCGC ATGCAT GGGGGCGTGGCGGAGAGC TCCACC CTAAGT CCATTC CACAGA TCAGCT GAGCAG AGCCGG CCTGAAGGCCAGCCCTTG AAATAC CCTTCA TGGTGA AAGCGC TAAGCT GTTTTA CGATTA TTAACTCCAGTCCTTGCC TTCTGC AGATGG CTCAAG TGAAAC GCCAGC TTTTTT GGTGAG CTAGAGAAGCAAGATGAT CATCTG TCAGAG GGCAAC TTGGGG GAGTTC TCTTCT ATCAAG AGTGTCCTGGGGATTGAA CTCAGT TCATCC ATCATT CTTCAT GTACCC GTTTTC TAAGCC AGTTTCTCAGCCACTTGA TTAGAA TCTTTC AAAAAA AAATTG TATGGA TATTTT GTCCAA ATGTATTTTCCGTGCATCATGTTT ACGCAA TTGCCT GTAGAG ACCAGA AGAGGA TATTAA GTCCCAGATGTCCAGTGG AACTGG GATCAC AGATGT TTGTGG GCTGTT GCGTTG TGGTGC TTGGAACTGAACCTGGGT CTTCTG CAAGAG CAGCAA GCATTC TTAACT ACTGAG CCATCT TGTCAGCCTAAAACTTTG TTTTTG TTTTTT TGTTTT TGTTTT TAATCA CACCTT TAGGGG TTGGGAATGTAACTCAGT TGGTAG TAGAGT GGGTGA AGTGCA TGAAGC TCTCTC TCTGGG TTTGATCCCTAGTGATAT GTAAGC CAGGCT TGGTGG TACATC ACTGCA GTCCTG GCACTT GAGATGTGGAGGTATGAT CAGTTC GAGATT ATCCTA ATCCAT AGGAAG CTTGAG GCTAGC TTGCGCTAGGGATACTCT CATAAT AAATAG ATAAAT AAATAA AGTCAC ATAGGA GAACCA TACACAACTCTTAAGTAT CTGGGT CACTCA GCTTTT TGTTTC TATGGA AACCTC CCTGGG ACAAAGCAGGAGTGGTTT ATTTTG TCTCAT AGTTTC AAAGGG TCAGTC CATCGT TGGCCA AAAGCTCTGGGGTTTTTA GCTTGA GCACAC AATGCA ATTTAA TTGGGT ATCTCT GCCTGT GGGTTCCCCACCCACCTG CCCAGA GGGGGC TCCAGC TATTTC TGCCTA GGCTTA CATTGT TTCTTTCTTCCTCCGTGT GCCTTA TGCTGA TTTGTG AGTCTC AGGCAT GGATTG(SEQ ID NO.4)

[0039] The second homologous arm

[0040] TGTTCG GACAGA ATACCT TGGCAG CAGGAG CATATA GTGGAA GTGAGC TGTTGC TAGCCTTGTAGG GGTCAA GAAACA GAAAAG GAGAAG ACAGGA AGAGGC TGGGAC AAGATA GAGCAT CTAAGCACGCCCATTG TGACTTCCTCA CCTTCA GCTTCA AATGTC CTCATATGATAA ATGCTT CAAAGG ATTCAT CTATTC ATCATC AGGTCA GAGCCC TCATGA TCTAACAGTTTCTGGAAA TGCCCC TGTAGA CACACC CACAG TGTGTT CCACTA TCCCAT TAGGTATCTGTC CACCGAGATCCAATT GAGGAGATCCAATT ATTG AGCACA GAGGGTGTGGCC ACCAGC CAGGTACATGCT ATGGGG TCACGG TCTCAA GCTTCA GGATAT CTGAAGACTTAT TATTAT TGTTAT TTCGGGAAGCTC TAGGGG TGCAGC TTTCAT GCCCGG ACAGACTGACTGACCTT GGCTTGCT CGCTTGTT ATGGGG GTGGAC ATCCCAGTTAAG TCCCTAA GAGTGA ATTGTG ATGGTTTCCTCA GGTCAG AGGTAGCCACA GAGCTG CAAACT TGACTC AGATCA GCAGAGTTGGGT GTATGG CCTTAGTCCTAG AGTGGCAA TGTACAA AACCAGACAGGC AGATGACAAAGC AAGCCA GTCTGT GCTATA AGCTAC CCAGTT CTTCCT ATCAGA ATCATGGTTGTAAAAGAG CCTCAT TTCTCA TCAGCG GCATTA ACCCAC ATCACC GCAGTG ACACACTGATTGTGAAGT GCAAGGCAG ACTCAGCTATGC CACGGG CTATGT CCAGGC ATGAAAGCTGCATCCCTT GAGCTT CCTGCA CAGCCT GCACAC ACGATC ATGTAT GCAGCA GCTCTGTGAGTAGGTGTT TCTTCC CCACAG AAGTTG TGATCT TGTGAA ACTTGT TTTACT ATATTACAGAAGAGTATT TACTGG GGGAGG GTGATG CTTGCA CACACA GCACAG TGCACA CGTGGAGGTCAGAGGACA ACTTGA AGGAGT TGATTC TCTCCT TCTATC CTGTGG GTCCTG AGGGTCAAACTCAGGGTT TCAGGC TTGGTG GCACAC ACTGTG TTATGA GTCTAC TCAGTG TTCCATGGTGGATGCTAT GTTCTT GTAATA CTTTCT ATAGAC CTAATT TTTTTT CTTTTC TGTTTTATTTTTTAAAA TTTATT TATTTA TTTCAT GTATGT GAGTAC ACTGTT GCTATC TTCAGACACAGAAGA GGGCAT TGGATC CCCATT ACAGAT GGTTGT GAGCCA CCATGT GGTTGCTGGAATTGAAC TCAGGA CCTCTG GAAGAG CAGTCA GTGTTC TTAACC ACTGAG CCATCTCTCCAGCCCACT GTTTTT TGGTTG TTTTTG TTGTTG TTGTTT TTAAAT GAATGA GATACTTTAGCCCATAGA AGAAAG TCTATT AAATTA TTAAAT ATTCTG TGTATT TGGTTT TTACCCCTTTATGAGCAC TAAGAA AATAAA TGCTTA TTCAGA GAAAAT AGGTAG ATAAGG ATAGCCAGTAGTTTCTGA CTCATG CCTTGA AGTCCC TCCTTT GTGATA GAAGCA TACGTG TGTTTGTTTTTTATCCC TCTGCT GGATAT TCTGTG TCAACT TGACAC AGGCTA AAGTCA TCTCAGGAGAGAAGACCTCGATTG TGAACA TTGCTC CATAAA ATAGGA CTATAG GCAAGA CTGTTGGACATTTTCTTA ATTAGT GATGGA TGTGGG AGGGCC CAGACC ATTGTG GGTGGT GCGATCTCTGTCCTGGGT TCTATA AGAAAG CAGGCT GAGCAA GCTATGCAAGGACCAGGCCTAGCCACCTA TGGCCT CTGCAT CAGCTT CTGCCT CCAAGT TCCTGT CCAGTT TGACTTCCTGTCCAGACT TCCTCC AATGCT GAACAG CGATGT GGAAAT GTAAGC CAGTAA ACCCTTGCTTTGGTTGGT ATTTCA TCATAG CAATGC AAACCT TGACTT AGGCCCTA GCCTAGATTGATTGATTGATTGATT TAGATC TATTCT TCCATT ACTTGC TGCTGG TATTGA ATGTCAGCACAGGGACTA TGGTAA GACAGC TTTCTT TTCATA CTGTTT TGAAGT CAAAGT CTTAAAGTGATAAGACAT TTTTTT CCTCAT ACTTGT TTTAAT AGTAAG GCTTAG ACTTAC ATCCATCATTGATCTAGTCTT CCAGAG ACTATT ACTCTA GGACAA TTGACT CTGTTTCTACATGCATTT TCTTTT ATTATG TTTATT TATTTT ATGGGC ATGAGT ATTTTG TTTGCATGTACTTATGCA CAATAC GTAAGT GCAGTG CCTAAG GAAGCC AGAAGA AGGTAT TGACCGATTGATCAGATGATGTTG CTGTGA GCTCCT GTGTGA ATGCTG GGGCCC AAACTAAGGTCCTCTGCA AGTGCC TTCGGT ACTCTT AACCTT TGAGCC ATCCCT CCAGTC TACACACTTTCATGAAGC TTCCAC TACCCC ACCTTA GCTCCA GGAACT AGCAGC ACTACCAGCCCTGACCCAGCTTCC TGTGAA TCCATG GATAAA AGACAC ACAGAC ACACAC AGGTTG CTCATTTTCAACTAGCCT TCTGAC ACAATT GCTGGG TGCTGC TATCTC CCGTCT GGAAAA GTGTGCCCTTATCAGTAT TCTTAG CTCTGT ACCTCC CACCTG CCCTAA ACTTTA GTTGAT CAGTTACATCTAGTCCCT GTTAAC ATCCCT GACAGA CACACA GGCTGC CACTCC TCCCTC GATCTCACATGGCTTCTT GGCTTT TCTCTC TCTGAA GCATGG TGAACT CCTCCCTT(SEQ ID NO.5)

[0041] In a specific embodiment, the nucleic acid molecule comprises the following elements: DTA-first homology arm-LoxP-target sequence-SDA-Neo-SDA-LoxP-second homology arm.

[0042] Preferably, the RAB31 inhibitor is a nucleic acid construct (targeting vector), and the nucleic acid construct comprises the nucleic acid molecule described above.

[0043] More preferably, the method for constructing the nucleic acid construct is: inserting two LoxP sites in the same direction on both sides of the second exon of RAB31. Specifically, using BAC clone RP24-36513 as a template, PCR is used to generate the first homology arm, the second homology arm and the second exon (cKO), that is, the first homology arm-target sequence-second homology arm. Two LoxP sites in the same direction are connected on both sides of the second exon of the RAB31 gene (first homology arm-LoxP-target sequence-LoxP-second homology arm), and connected to the first homology arm (first homology arm-LoxP-target sequence-LoxP); the Neo screening marker is connected between two self-deletion anchors (SDA), and the 5' end is connected to the target sequence; the DTA screening marker is connected to the upstream of the first homology arm. The sequence of the nucleic acid construct is shown in SEQ ID No.6.

[0044] The PCR primer sequences used are:

[0045] F1: 5'-TCCCTTATATCAGCGAAACAGCCA-3' (SEQ ID No. 7)

[0046] R1: 5'-AGCAAGCTTCCCAGACAGTC-3' (SEQ ID No. 8)

[0047] Preferably, the RAB31 inhibitor is a gene editing system, and the gene editing system is selected from one or more of the Cre-Loxp system, the Flp-FRT system, the Dre-ROX system, the vCre-vloxp system, the sCre-sloxp system, the CRISPR / Cas9 system, the CRISPR / Cas12 system, and the CRISPR / Cas derived single base editing system. The gene editing system uses the sequence shown in SEQ ID No.1 as the target sequence (knockout region or cKO region) for knockout. In certain specific embodiments, the RAB31 inhibitor is a Cre-Loxp system, and the Cre-Loxp system comprises the nucleic acid construct described above, and the nucleic acid construct knocks out the RAB31 gene under the action of Cre recombinase.

[0048] Preferably, the RAB31 is used alone or in combination with other drugs. That is, the RAB31 inhibitor can be used as 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 in the form of common solid, liquid, gel, semi-fluid, aerosol, etc.

[0049] Preferably, the product is mainly targeted at mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0050] Preferably, the RAB31 inhibitor prevents and / or treats hypertension through at least one of the following:

[0051] 1) Improve decreased vascular function;

[0052] 2) Improve reduced heart function;

[0053] 3) Improve blood vessel wall thickening.

[0054] More preferably, the reduced vascular function refers to an increase in the vascular pulse wave propagation velocity, the improved reduced vascular function refers to a reduction in the vascular pulse wave propagation velocity, and the reduction in the vascular pulse wave propagation velocity refers to bringing the vascular pulse wave propagation velocity of the target vessel close to or reaching the velocity of a healthy vessel. For example, based on a healthy vessel, the product can restore the vascular pulse propagation velocity of the target vessel to at least 70%, 80%, 90% or 100% of that of a healthy vessel.

[0055] More preferably, the reduced cardiac function refers to a reduced cardiac ejection fraction or a reduced left ventricular fractional shortening, and the improved reduced cardiac function refers to an increased cardiac ejection fraction or a reduced left ventricular fractional shortening, and the increased cardiac ejection fraction or a reduced left ventricular fractional shortening refers to bringing the cardiac ejection fraction and the left ventricular fractional shortening of the subject close to or reaching those of a healthy heart. For example, based on a healthy heart, the product can restore the cardiac ejection fraction and the left ventricular fractional shortening of the subject's heart to at least 70%, 80%, 90% or 100% of those of a healthy heart.

[0056] More preferably, the thickening of the vascular wall refers to the thickening of the vascular wall of the abdominal aorta, the improvement of the thickening of the vascular wall refers to reducing the thickness of the vascular wall, and the reduction of the thickness of the vascular wall refers to making the vascular wall of the target vessel close to or reaching the thickness of a healthy vessel. For example, based on a healthy vessel, the product can make the vascular wall thickness of the target vessel not exceed 130%, 120%, 110% or 100% of the thickness of a healthy vessel.

[0057] Another aspect of the present invention also protects a nucleic acid molecule or a substance related to a nucleic acid molecule for the preparation of a method for preventing or treating hypertension, wherein the target sequence of the nucleic acid molecule comprises the sequence shown in SEQ ID NO.1.

[0058] Preferably, the nucleic acid molecule comprises the following elements: LoxP-target sequence-LoxP. More preferably, the nucleic acid molecule further comprises one or both of a screening marker and SDA. The screening marker is selected from a positive screening marker and a negative screening marker, the positive screening marker is Neo, and the negative screening marker is DTA. Further preferably, the nucleic acid molecule further comprises a first homology arm and a second homology arm. The first homology arm is the first intron of RAB31, and the second homology arm is the second intron of RAB31. Specifically, the nucleic acid molecule comprises the following elements: DTA-first homology arm-LoxP-target sequence-SDA-Neo-SDA-LoxP-second homology arm.

[0059] Preferably, the substance includes one or more of the following: 1) a nucleic acid construct, which comprises the nucleic acid molecule as described above; 2) a gene editing system, which targets the sequence shown in SEQ ID NO.1; or, contains the nucleic acid construct described in 1); 3) a cell line, which comprises the nucleic acid molecule as described above; or, contains the nucleic acid construct described in 1); or, the cell line is obtained by transfecting a host cell with the gene editing system described in 2); 4) a pharmaceutical composition, which comprises the nucleic acid construct described in 1); or, comprises the gene editing system described in 2); or, the cell line described in 3).

[0060] More preferably, the nucleic acid construct can be obtained by cloning a gene fragment encoding the target sequence described above into a known vector. The known vector is a bacterial artificial chromosome BAC. Those skilled in the art can construct and obtain it by conventional methods of targeting expression vectors. For example, the target sequence is inserted into a plasmid containing Loxp, Neo and DTA by homologous recombination to form the nucleic acid construct. The nucleic acid construct comprises DTA-first homology arm-LoxP-target sequence-SDA-Neo-SDA-LoxP-second homology arm, and the sequence of the formed nucleic acid construct is as shown in SEQ ID NO.6.

[0061] Preferably, the gene editing system is a Cre-Loxp system, and the Cre-Loxp system comprises the nucleic acid construct (targeting vector) described above.

[0062] Preferably, the cell line refers to ES cells as host cells, and the gene editing system is transfected to knock out the RAB31 gene in the host cell. The ES cells are C57BL / 6NES cells. For example, the targeting vector in the gene editing system is linearized by enzyme cutting, and then the host cell is transfected by electroporation.

[0063] Further preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant or excipient, wherein the pharmaceutically acceptable carrier refers to a carrier that does not induce an allergic reaction or other discomfort in the patient to which it is administered. The excipient includes one or more of the following: a pharmaceutically acceptable solvent, a dispersant, an additive, a plasticizer, etc. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

[0064] Another aspect of the present invention also protects the use of a RAB31 inhibitor in preparing a kit for reducing the expression of the RAB31 gene.

[0065] The inventors of the present invention have found that the RAB31 inhibitor can effectively remove the second exon on RAB31, so that the RAB31 gene loses its function and is not expressed, and can be used to prepare a kit for reducing the expression of the RAB31 gene.

[0066] Another aspect of the present invention also protects a method for screening drugs for preventing and / or treating hypertension, the method comprising: taking RAB31 as a drug target, and searching for substances that can inhibit or block the expression and / or function of RAB31 as candidate drugs.

[0067] Preferably, an in vitro cell model or animal model with overexpression of the RAB31 gene is constructed, the drug to be selected is applied to the in vitro cell model or animal model, and then the content of RAB31 is detected. The cells may be from mammals.

[0068] The experimenter can determine whether the candidate drug is a drug with therapeutic significance by detecting the content of RAB31. Generally speaking, compared with the control group, a drug that can reduce the content of RAB31 by 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% can be determined as a drug with therapeutic significance.

[0069] Another aspect of the present invention also protects a method of treatment, comprising: administering a therapeutically effective amount of a RAB31 inhibitor or a substance related to a nucleic acid molecule to an individual. The method of treatment provided by the present invention can be used to treat indications including but not limited to hypertension. The therapeutically effective amount generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period. The individuals generally include humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which can benefit from treatment using the preparation, kit or combination preparation. The following specific embodiments illustrate the embodiments of the present invention, and those familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0070] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0071] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0072] Example 1

[0073] In this example, the heart rate, abdominal aortic media thickness, pulse wave velocity, ventricular ejection fraction, left ventricular short axis shortening rate and vascular pathological changes of mice after RAB31 gene knockout were investigated. They include the following:

[0074] 1. Experimental Materials

[0075] 1.1 Experimental drugs and main reagents

[0076] Angiotensin II (AngII, batch number Ab12018), Abcam, USA; isoflurane (batch number 970-00026-00), Shenzhen Ruiwode Company; anhydrous ethanol (batch number 1280340101602), Xilong Scientific Co., Ltd.; xylene (batch number 1430030101600), Xilong Scientific Co., Ltd.; eosin staining solution (batch number G1100), hematoxylin staining solution (batch number G114), Beijing Solebow Company; paraformaldehyde (batch number LA0427), Fuzhou Feijing Biotechnology Co., Ltd.; implantable capsule osmotic pump (batch number 2002D), Alzet Company, USA.

[0077] 1.2 Main instruments of the experiment

[0078] Pipette (Raining, USA); electronic balance scale (Ohaus Instrument Co., Ltd., Shanghai); non-invasive rat tail sphygmomanometer (Kent Co., Ltd., USA); small animal ultrasound imaging system Vevo2100 (Fujifilm Investment Co., Ltd.); inhalation small animal anesthesia machine (Shenzhen Ruiwode Life Science Technology Co., Ltd.); automatic biological tissue dehydration machine (Hubei Xiaogan Yaguang Medical Electronic Technology Co., Ltd.); paraffin embedding machine (Hubei Xiaogan Yaguang Medical Electronic Technology Co., Ltd.); pathological slicer (Leica, Germany).

[0079] 1.3. Construction and grouping of experimental animal models

[0080] 1.3.1 Construction of RAB31 knockout C57BL / 6 mice

[0081] By querying the expression characteristics of the RAB31 gene, designing a reasonable knockout region, constructing a targeting vector, electroporating ES cells, screening positive correct clones, breeding and identifying surrogate mice, and finally obtaining RAB31 knockout mice. The overall strategy is shown in Figure 1 .

[0082] 1) Construction of targeting vector

[0083] The RAB31 gene number in the NCBI database is NM_133685; the number in the Ensembl database is ENSMUSG00000056515, and it is located on mouse chromosome 7; a total of 7 exons were identified, starting from the start codon ATG of exon 1 and ending with the stop codon TGA of exon 7 (Transcript: ENSMUST00000070673).

[0084] Exon 2 was selected as the conditional knockout region (cKO region), the deletion of which should result in loss of function of the mouse RAB31 gene.

[0085] The sequence of exon 2 is shown in SEQ ID No.1.

[0086] The homology arms and conditional knockout region (cKO region) of the RAB31 gene were amplified by PCR using high-fidelity Taq DNA polymerase and BAC clone RP24-36513 as a template. The PCR primer sequences used were:

[0087] F1: 5'-TCCCTTATATCAGCGAAACAGCCA-3' (SEQ ID No. 7)

[0088] R1: 5'-AGCAAGCTTCCCAGACAGTC-3' (SEQ ID No. 8)

[0089] The nucleic acid molecule generated by PCR comprises the following elements: a first homology arm - exon 2 and a second homology arm.

[0090] The process of constructing a targeting vector is: the amplified product obtained by PCR amplification is sequentially connected with the recombination site and the selection marker to assemble into a targeting vector. Specifically, two loxP sites leading to the second exon of the mouse RAB31 gene are inserted on both sides, and two SDA (self-deletion anchor) sites and Neo positive screening marker elements are inserted, and finally the DTA negative screening marker element is inserted at the 5' end of the 5' homology arm. The targeting vector contains the following elements: DTA-first homology arm-LoxP-exon 2-SDA-Neo-SDA-Loxp-second homology arm. Neo is a neomycin resistance gene, which serves as a positive selection marker; DTA is a toxin gene, which serves as a negative screening marker; SDA is a self-deletion anchor.

[0091] The LoxP sequence is shown in SEQ ID NO.2, the SDA sequence is shown in SEQ ID NO.3, the first homology arm is shown in SEQ ID NO.4, the second homology arm is shown in SEQ ID NO.5, and the sequence of the targeting vector is shown in SEQ ID No.6.

[0092] Figure 1 This is a strategy diagram for constructing a targeting vector, where Wildtype allet is the allele of RAB31, Targeting vector is the targeting vector, Mutant allele 1 is mutant allele 1 (Targeted allele), Mutant allele 2 is mutant allele 2 (After Neo deletion), Mutant allele 3 is mutant allele 3 (After Cre deletion), Exon is exon, loxp site is loxp site, cKO region is knockout region, SDA is self-deletion anchor, and Homology arm is homology arm.

[0093] The targeting vector is linearized using an enzyme. The schematic diagram of the linearized targeting vector is shown in Figure 2 .

[0094] 1.2. Electroporation and screening of targeting vectors: The linearized targeting vectors are introduced into mouse ES cells by electroporation. After overnight culture, positive and negative screening is performed, and positive recombinant ES cells are retained for injection into the blastocyst cavity of recipient mice.

[0095] 1.3. Blastocyst injection: The positive cell clones that have undergone homologous recombination are injected into the 3.5-day blastocysts of C57BL / 6N donor mice, and the blastocysts are transplanted into the uterus of CD-1 pseudo-pregnant mice to produce F0 generation positive chimeric mice.

[0096] 1.4. Obtaining homozygous offspring: F0 positive mice were bred with tissue-specific Cre mice (WT wild-type mice with cre enzyme, Saiye Biotechnology Co., Ltd.) at a rate of 1♂ and 2♀Cre to obtain F1 mice (flox / + ,Cre) and F1 mice (flox / + ), identification; F1 generation mice (flox / + ,Cre) and F1 mice (flox / + )) 1♂×2♀ breeding, obtain F2 generation mice (flox / flox ,Cre) and F2 mice (flox / flox ), F2 generation mice (flox / flox ) is also Rab31 - / - (KO) mice were raised to 8-10 weeks of age for the next experiment.

[0097] flox / + : Indicates that the locus is heterozygous, that is, one allele has a flox site and the other is a wild-type allele. / flox : Indicates that the locus is double flox, with both alleles carrying flox sites.

[0098] 1.3.2 Grouping

[0099] Take 8-10 week old male Rab31 + / + (WT) mice were randomly divided into 2 groups, WT+Saline group and WT+AngII group, with 6 mice in each group. The WT+AngII group (WT&AngII+) refers to WT mice subcutaneously implanted with a pre-placed osmotic minipump containing AngII; the WT+Saline group (WT&AngII-) refers to WT mice subcutaneously implanted with a pre-placed osmotic minipump containing saline.

[0100] At the same time, 8-10 week old male Rab31 - / - (KO) mice were randomly divided into 2 groups, KO+Saline group and KO+AngII group, with 6 mice in each group. Among them, KO+AngII group (Rab31 KO&AngII+) refers to KO mice subcutaneously implanted with pre-placed osmotic minipumps containing AngII; KO+Sham group (Rab31 KO&AngII-) refers to KO mice subcutaneously implanted with pre-placed osmotic minipumps containing saline.

[0101] The specific operation of osmotic micropump implantation was as follows: the day before surgery, the osmotic micropump was taken out in the clean bench, and the osmotic micropumps in the AngII group were injected with 200 μL of AngII solution (500 ng / kg / min), and the Saline group was injected with 200 μL of normal saline solution, and all osmotic micropumps were immersed in normal saline in a cell culture incubator at 37°C overnight. On the day of surgery, the operating table was routinely disinfected, the mice were anesthetized with isoflurane, placed in a prone position, the hair on the back of the head and neck was removed, disinfected with iodine, the skin was cut open with scissors, the subcutaneous tissue was bluntly separated, and the mice in the AngII group were subcutaneously implanted with pre-placed osmotic micropumps containing AngII, and the mice in the Saline group were subcutaneously implanted with pre-placed osmotic micropumps containing normal saline, and then the skin was sutured and disinfected.

[0102] 2. Performance testing

[0103] 2.1 Blood pressure measurement

[0104] In this experiment, a non-invasive rat tail artery sphygmomanometer was used to monitor the blood pressure of each group of mice. The blood pressure of experimental mice was monitored by the Kent non-invasive blood pressure instrument in the United States. Before blood pressure monitoring, the mouse was placed in a restraint, and an occluder and a volume pressure sensor were put on the part about 1 cm away from the root of the tail. Then it was placed on a heating plate, and the appropriate temperature was selected and left to stand for 10 minutes to keep the mouse in a resting state. When the mouse was in a calm state, blood pressure measurement was started. The occlusion sleeve and VPR sensor were inflated and deflated 15 times in a cycle, and the computer software was set to measure 15 times and take the average value. The instrument can measure the systolic pressure, diastolic pressure and mean arterial pressure of the tail artery of small animals.

[0105] 2.2. Measurement of abdominal aorta pulse wave propagation velocity and vascular wall thickness

[0106] Vevo 2100 animal ultrasound was used to detect the pulse wave propagation velocity and media thickness of the abdominal aorta of mice. Before the test, the mice were anesthetized by inhalation of isoflurane, the left chest and abdomen were depilated, and the mice were fixed in a supine position on a 37°C heating plate. Coupling agent was applied to the abdomen and ultrasound probe of the mice, and then the probe was placed parallel to the middle of the abdomen. B-mode ultrasound was used to obtain the abdominal aorta and the probe was placed perpendicular to the midline of the abdomen. M-mode was used to record the conduction of the proximal and distal ends. The pulse propagation velocity of the mice was calculated by measuring the data of the abdominal aorta vascular length and the terminal delay time. The parasternal long axis section of the mice was further collected, and B-mode sampling was performed and the image was saved. On the long axis section, the image was enlarged, and M-mode sampling was performed and the image was saved. Three cardiac cycles were recorded continuously for each mouse, and the cardiac function indexes could be calculated based on the measurement results.

[0107] 2.3 HE staining

[0108] When sampling, the abdominal aorta of mice was dissected, fixed in 4% paraformaldehyde for 24 hours, and then dehydrated with gradient ethanol of different concentrations, immersed in xylene to replace ethanol, and immersed in wax. The tissue blocks were switched into 4 μm slices by a slicer, spread in 37°C water, transferred to a slide, and baked, dried, hydrated in xylene, dewaxed with gradient ethanol of different concentrations, stained with hematoxylin for 70 seconds, rinsed with tap water, differentiated with 1% hydrochloric acid for 1 second, placed in water for anti-blueing for 10 minutes, placed in eosin solution for staining for 2 seconds, dried, sealed, and the pathological morphological changes of the tissues were observed under a microscope.

[0109] 2.4 Statistical Analysis

[0110] First, the Shapiro-Wilk test was used to analyze whether the data conformed to the normal distribution, and the variance homogeneity test was performed for the data with normal distribution. For data that conformed to the normal distribution and had homogeneous variance, ANOVA multiple group comparisons and Bonferroni test were used; for data that conformed to the normal distribution but had unequal variance, ANOVA multiple group comparisons and Kruskal-Wallis test were used. For data that did not conform to the normal distribution, nonparametric tests were used. For data with repeated measurements in multiple groups, repeated measurement data sphericity test and variance analysis were used. P<0.05 was considered statistically significant. All data analyses were performed in SPSS26.0.

[0111] 3. Experimental results

[0112] 3.1. RAB31 knockout alleviates the increase in blood pressure in mice after Ang II stimulation

[0113] The effect of RAB31 knockout on systolic blood pressure in mice was detected by non-invasive tail artery blood pressure monitoring. Figure 3 ), diastolic blood pressure( Figure 4 ) and mean arterial pressure ( Figure 5 )’s impact.

[0114] from Figure 3 , 4 As shown in Figures 5 and 5, before surgery, there was no statistically significant difference in blood pressure among the four groups of mice (P>0.05).

[0115] from Figure 3 , 4 As shown in Figures 5, the tail artery blood pressure of mice was measured at 1, 2, 3, and 4 weeks after surgery. Compared with the WT+Saline group, the systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) of the WT+AngII group were significantly increased, and the difference was statistically significant (P < 0.05). Compared with the WT+AngII group, the systolic blood pressure, diastolic blood pressure and mean arterial pressure of the KO+Saline group and the KO+AngII group were significantly decreased, and the difference was statistically significant (P < 0.05), indicating that knocking out RAB31 significantly inhibited the increase in blood pressure.

[0116] 3.2. Rab31 knockout reduces the thickness of the tunica media and pulse wave propagation velocity of the abdominal aorta of mice after AngII stimulation

[0117] After the experiment, the abdominal aorta media thickness and pulse wave propagation velocity of each group of mice were measured using a small animal ultrasound instrument. The results are as follows: Figure 6 shown.

[0118] from Figure 6 It can be seen that compared with the WT+Saline group, the media thickness and pulse wave propagation velocity of the abdominal aorta in the WT+AngⅡ group were significantly increased; compared with the WT+AngⅡ group, the media thickness and pulse wave propagation velocity of the KO+AngⅡ group were significantly reduced, and the difference was statistically significant (P<0.05). The above results suggest that RAB31 knockout significantly inhibits the increase of the media thickness and pulse wave propagation velocity of the abdominal aorta after AngII stimulation.

[0119] 3.3 Effects of RAB31 knockout on vascular pathological morphology in mice after Ang II stimulation

[0120] HE staining was used to compare the pathological changes of the abdominal aorta of each group of mice. Figure 7 shown.

[0121] from Figure 7 It can be seen that compared with the WT+Saline group, the abdominal aorta of mice in the WT+AngII group was significantly thickened, while compared with the WT+AngII group, the thickness of the abdominal aorta of mice in the KO+AngII group was significantly reduced. This result suggests that RAB31 knockout can alleviate vascular pathological changes after AngII stimulation.

[0122] 3.4. RAB31 knockout improves cardiac contractile function in mice after AngII stimulation

[0123] The cardiac contractile function of each group of mice was detected by small animal ultrasound. Figure 8 shown.

[0124] from Figure 8 It can be seen that compared with the WT+Saline group, the left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS) of mice in the WT+AngⅡ group were significantly reduced; compared with the WT+AngⅡ group, the left ventricular ejection fraction and left ventricular short axis shortening rate of mice in the KO+AngⅡ group were significantly increased, and the difference was statistically significant (P<0.05). The above results suggest that RAB31 knockout significantly inhibits the cardiac contractile dysfunction of mice after AngII stimulation.

[0125] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. The use of RAB31 gene as a target in screening products for the prevention and treatment of hypertension.

2. Use of a RAB31 inhibitor in the preparation of a product for the prevention or treatment of hypertension, wherein the RAB31 inhibitor is one or more selected from the following: 1) a nucleic acid construct, the nucleic acid construct It includes the following elements: DTA-first homology arm-LoxP-exon 2-SDA-Neo-SDA-Loxp-second homology arm; The Neo is a neomycin resistance gene, which is used as a positive selection marker; the DTA is a toxin gene, which is used as a negative selection marker; and the SDA is a self-deletion anchor point; The exon 2 is as shown in SEQ ID NO.1; the LoxP sequence is as shown in SEQ ID NO.2; the SDA sequence is as shown in SEQ ID NO.3; the first homology arm is as shown in SEQ ID NO.4; the second homology arm is as shown in SEQ ID NO.5; the sequence of the nucleic acid construct is as shown in SEQ ID NO.6; 2) Gene editing system: the gene editing system comprises the nucleic acid construct described in 1), and the gene editing system is selected from the Cre-Loxp system; 3) Cell line: the cell line contains the nucleic acid construct described in 1), or the cell line is obtained by transfecting a host cell with the gene editing system described in 2); 4) A pharmaceutical composition, comprising: 1) the nucleic acid construct; or, 2) the gene editing system; or, 3) the cell line.

3. The use according to claim 2, characterized in that The RAB31 inhibitor has at least one of the following effects: 1) Improving decreased vascular function, wherein the improvement of decreased vascular function refers to reducing the propagation velocity of vascular pulse waves; 2) Improving reduced cardiac function, wherein improving reduced cardiac function refers to increasing cardiac ejection fraction or left ventricular shortening fraction; 3) Improving vascular wall thickening, wherein improving vascular wall thickening refers to reducing the thickness of the vascular wall.

4. The use according to claim 2, characterized in that The RAB31 inhibitor is the only active ingredient of the product.

5. The use according to claim 2, characterized in that The RAB31 inhibitor is one of the active ingredients of the product.

6. A method for screening drugs for preventing and / or treating hypertension, characterized in that: The method comprises: taking RAB31 as a drug target, and searching for a substance capable of inhibiting or blocking the expression and / or function of RAB31 as a candidate drug.

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