MiR-92a-1-5p high expression vector for improving cardiac fibrosis and fibroblast line

Abstract

The invention discloses a miR-92a-1-5p high expression vector for improving cardiac fibrosis and a fibroblast cell line. The invention provides a DNA fragment for expressing miR-92a-5p, the sequence of the DNA fragment is shown as SEQ ID NO: 4, the invention also provides an expression vector containing a target gene sequence shown as SEQ ID NO: 4 and a human heart fibroblast cell line with high expression of miR-92a-1-5p, and the cell line can improve the fiber activation capability of human heart fibroblasts, inhibit the migration capability of the fibroblasts, and improve the human heart fibroblasts. And the collagen shrinkage capability of fibroblasts is inhibited. In addition, the miR-92a-1-5p overexpression recombinant vector constructed by the invention is helpful for exploring the molecular mechanism of the miR-92a-1-5p in the occurrence and development of fibrosis diseases such as fibrosis after myocardial infarction, and is helpful for developing a therapeutic scheme of the fibrosis diseases; the method has an important application value.

Description

Technical Field

[0001] The invention relates to a miR-92a-1-5p high expression vector and a fibroblast cell line for improving cardiac fibrosis, belonging to the technical field of molecular biology and genetic engineering. Background Art

[0002] Cardiovascular diseases have a high morbidity and mortality rate worldwide. Myocardial infarction is an acute cardiovascular event. If it is not treated with timely reperfusion, it often leads to fatal arrhythmias, heart failure and other adverse prognoses. Myocardial fibrosis plays a vital role in the process of ventricular remodeling after myocardial infarction. In the early stage of infarction, it promotes scar repair through fibroblast activation and maintains the integrity of the damaged ventricular wall structure and function. However, excessive fibrosis in the later stage will lead to a large amount of collagen aggregation, hindering the heart's contractile function and causing ventricular dilatation. The progression of cardiac fibrosis after myocardial infarction is accompanied by several significant features, including the proliferation of cardiac fibroblasts, the transformation of fibroblasts into myofibroblasts, and excessive extracellular matrix deposition.

[0003] The TGF-β / Smad signaling pathway plays a key role in the regulation of fibroblasts and the progression of fibrosis. In the infarcted area and infarct margin area after myocardial infarction, active TGF-β binds to and phosphorylates type I and type II serine / threonine transmembrane receptors TGFBR2 and TGFBR1 on the cell membrane, and responds to the phosphorylation of its downstream effector protein Smad family and different forms of combination, transmitting extracellular signals from the cell membrane to the nucleus. Active TGF-β binds to and phosphorylates TGF-β receptor 2 (TGFBR2) and TGF-β receptor 1 (TGFBR1). Phosphorylation of TGFBR1 activates the type I receptor kinase domain, and then propagates intracellular signals downstream through Smad proteins, enhancing the synthesis of extracellular matrix proteins and promoting post-infarction ventricular remodeling.

[0004] Effective treatment of myocardial fibrosis can block the process of ventricular remodeling, thereby improving cardiac function and infarction prognosis. However, the pathophysiological process and molecular mechanism of post-infarction fibrosis still need to be improved, and there is still a lack of safe and effective treatment methods in clinical practice. Therefore, it is crucial to discover new targets for the process of cardiac fibrosis, which will help improve anti-fibrosis treatment.

[0005] MicroRNA (miRNA) is a non-coding RNA with a length of about 20-25 nucleotides found in eukaryotic organisms. It can recognize target mRNA through complementary base pairing and guide its degradation or inhibit its translation. It plays a vital role in the occurrence and development of various diseases including cardiovascular diseases. Various miRNAs can be used as new early diagnostic indicators for cardiovascular diseases. Among them, the miR-17-92 gene cluster is highly conserved in mammals. It has a total of 6 members, namely miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a. In recent years, studies have found that members of the miR-17-92 gene cluster can regulate the occurrence and development of various cardiovascular diseases including atherosclerosis, heart failure, pulmonary hypertension, coronary heart disease, cardiomyopathy, etc. alone or in a coordinated manner. However, there are no reports on the role of miR-92a-1-5p in myocardial fibrosis. Summary of the invention

[0006] The present invention aims to construct a miR-92a-1-5p high expression vector and fibroblast cell line for improving cardiac fibrosis, and is used to study its effect on cardiac fibrosis. The results show that it has an activation effect on fibroblasts and has the effect of inhibiting the progression of fibrosis.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a nucleotide fragment expressing miR-92a-1-5p, the sequence of which is shown in SEQ ID NO:4.

[0009] In a second aspect, the present invention provides an expression vector, wherein the expression vector comprises a target gene sequence as shown in SEQ ID NO:4.

[0010] Preferably, the expression vector is selected from a virus, fungus or bacterial expression vector.

[0011] In a third aspect, the present invention provides a cell comprising the expression vector described in the second aspect, wherein the cell is a human cardiac fibroblast, and the human cardiac fibroblast is a commercial in vitro cell.

[0012] In a fourth aspect, the present invention provides a method for preparing a recombinant lentiviral expression vector, comprising: digesting the lentiviral vector pHBLV-U6-MCS-PGK-PURO with restriction endonucleases EcoRI and BamHI, and cloning the target sequence shown in SEQ ID NO: 4 into the digested lentiviral vector to obtain a recombinant lentiviral vector.

[0013] In a fifth aspect, the present invention provides a method for preparing a human cardiac fibroblast cell line with high expression of miR-92a-1-5p, comprising the following steps:

[0014] Step 1: The recombinant lentiviral vector is combined with the virus packaging auxiliary plasmids pSPAX2 and pMD2G to form a three-plasmid lentiviral system for virus packaging, and then transfected into 293T cells to obtain a virus liquid; the recombinant lentiviral vector is a recombinant lentiviral vector prepared by the preparation method according to claim 5;

[0015] Step 2: Infect human cardiac fibroblast cell lines with the virus solution obtained in step 1, and screen and obtain human cardiac fibroblast cell lines with high expression of miR-92a-1-5p, i.e., fibroblast cell lines with high expression of miR-92a-1-5p.

[0016] In a sixth aspect, the present invention provides the expression vector of the second aspect, and use of the recombinant lentiviral vector prepared by the preparation method of the fourth aspect in any one of the following A1)-A10):

[0017] A1) preparing a reagent for promoting the expression of miR-92a-1-5p;

[0018] A2) preparing a product for improving fiber activation of human cardiac fibroblasts;

[0019] A3) preparing a product for inhibiting the migration ability of human cardiac fibroblasts;

[0020] A4) preparing a product for inhibiting collagen contraction in human cardiac fibroblasts;

[0021] A5) preparing a product for constructing a cell model or animal model with high expression of miR-92a-1-5p;

[0022] A6) promoting miR-92a-1-5p expression in vitro;

[0023] A7) ability to improve fiber activation of human cardiac fibroblasts in vitro;

[0024] A8) Inhibits the migration ability of human cardiac fibroblasts in vitro;

[0025] A9) Inhibits the collagen contractility of human cardiac fibroblasts in vitro;

[0026] A10) Construct a cell model with high expression of miR-92a-1-5p.

[0027] In a seventh aspect, the present invention provides the cell described in the third aspect, and the use of the human cardiac fibroblast cell line with high expression of miR-92a-1-5p prepared by the preparation method described in the fifth aspect in any one of the following B1)-B7):

[0028] B1) preparing a product for improving fiber activation of human cardiac fibroblasts;

[0029] B2) preparing a product for inhibiting the migration ability of human cardiac fibroblasts;

[0030] B3) preparing a product for inhibiting collagen contraction in human cardiac fibroblasts;

[0031] B4) preparing a product for constructing an animal model with high expression of miR-92a-1-5p;

[0032] B5) ability to improve fiber activation of human cardiac fibroblasts in vitro;

[0033] B6) Inhibit the migration ability of human cardiac fibroblasts in vitro;

[0034] B7) Inhibits the collagen contractile ability of human cardiac fibroblasts in vitro.

[0035] B8) Study the molecular mechanism by which miR-92a-1-5p inhibits cardiac fibrosis.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention utilizes lentiviral vector overexpression technology to achieve overexpression of miR-92a-1-5p in human cardiac fibroblasts, and discovers for the first time that miR-92a-1-5p is a key molecule for improving cardiac fibrosis. High expression of miR-92a-1-5p can improve the ability of human cardiac fibroblasts (HCF) to activate fibers under TGF-β-induced fibrosis intervention conditions, inhibit the migration ability of fibroblasts, and inhibit the collagen contraction ability of fibroblasts. This will provide an effective tool for clarifying the association between miR-92a-1-5p and fibrosis in various pathological modeling environments in the future and for exploring the potential molecular mechanisms and signal networks between miR-92a-1-5p and fibrosis, greatly reducing the time and economic cost of early gene editing; and providing useful clues for the treatment of fibrotic diseases such as post-myocardial infarction fibrosis; therefore, the present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 For RT-qPCR identification of miR-92a-1-5p overexpression effect;

[0039] Figure 2 The effect of miR-92a-1-5p overexpression on the activation of human cardiac fibroblasts under TGF-β-induced fibrosis intervention conditions;

[0040] Figure 3The effect of miR-92a-1-5p overexpression on the migration of human cardiac fibroblasts under TGF-β-induced fibrosis intervention conditions;

[0041] Figure 4 This is the effect of miR-92a-1-5p overexpression on collagen contraction of human cardiac fibroblasts under TGF-β-induced fibrosis intervention conditions. DETAILED DESCRIPTION

[0042] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0043] The inventors found in a study that the miR-92a-1-5p levels in blood samples of patients after myocardial infarction showed differential expression as myocardial fibrosis continued to progress, suggesting that it was associated with the progression of fibrosis after myocardial infarction. On this basis, the inventors conducted research on miR-92a-1-5p, and thus completed the present invention.

[0044] In the present invention, the Targetscan database (www.targetscan.org) was used to query and obtain the nucleotide sequence of miR-92a-1-5p, as shown in SEQ ID NO: 1, and the full length of the sequence is 23 bp.

[0045] In order to make the miR-92a-1-5p expressed by the lentiviral expression vector completely identical in sequence and structure to the miR-92a-1-5p physiologically synthesized in the cell, the present invention adds a miRNA stem-loop fragment and a reverse complementary fragment lacking the 9th and 10th nucleotides on the basis of the miR-92a-1-5p nucleotide sequence, and its nucleotide sequence is 5'-TTCAAGAGA-3' and as shown in SEQ ID NO: 3.

[0046] Example 1: Design and synthesis of double-stranded DNA fragments corresponding to miR-92a-1-5p

[0047] 1. Design and synthesis of double-stranded DNA fragments expressing miR-92a-1-5p

[0048] The gene sequence of human miR-92a-1-5p was searched through the Targetscan database (www.targetscan.org), and the RNA sequence of miR-92a-1-5p (SEQ ID NO: 1) was obtained as follows: 5'-AGGUUGGGAUCGGUUGCAAUGCU-3'. U was further converted to T to construct the DNA sequence required for the vector (SEQ ID NO: 2) as follows: 5'-AGGTTGGGATCGGTTGCAATGCT-3'. In order to reflect the stem-loop structure of miR-92a-1-5p, the stem-loop sequence was designed: 5'-TTCAAGAGA-3', and the reverse complementary sequence with the 9th and 10th nucleotides missing (SEQ ID NO: 3): 5'-AGCATTGCCCGATCCCAACCT-3', and finally the vector sequence of miR-92a-1-5p was constructed as follows (SEQ ID NO: 4):

[0049] 5'-GATCCGAGGTTGGGATCGGTTGCAATGCTTTCAAGAGAAGCATTGCCCGATCCCAACCTTTTTTTG-3', and synthesize the DNA template strand and the reverse complementary strand. Finally, according to the reaction system of 2μl 10*oligo Buffer, 1μl DNA template strand, 1μl reverse complementary strand, 16μl ddH2O, the template strand and complementary strand of the miR-92a-1-5p target fragment were annealed to form a target fragment DNA double strand according to the annealing program of 95℃10min, 75℃10min, 55℃10min, 35℃10min, 15℃10min.

[0050] 2. PCR reaction was used to amplify the target fragment of miR-92a-1-5p.

[0051] According to the sequence of the double-stranded DNA of miR-92a-1-5p, upstream and downstream primers with sticky ends were designed to facilitate the subsequent connection of the target fragment with the vector. The designed and synthesized upstream primer sequence is (SEQ ID NO: 5): 5'-GACGAGGATCCGAGGTTGG-3, and the downstream primer is (SEQ ID NO: 6) 5'-AGAATTCAAAAAAAGGTT-3'.

[0052] Prepare PCR amplification system: 25μL 2X PCR Buffer, 1μL dNTPs mix (10mM each), 2μL forward primer (10μM), 2μL reverse primer (10μM), 1μL cDNA template, 18μL ddH2O, 1μL phanta Super-FidelityDNA Polymerase. Amplify according to the PCR reaction conditions of pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 55℃~72℃ for 30s, extension at 72℃ for 30~60s / kb (27-35 cycles), and complete extension at 72℃ for 10min to obtain the miR-92a-1-5p target fragment with sticky ends.

[0053] Example 2: Construction of miR-92a-1-5p overexpression lentiviral vector and identification of its overexpression effect

[0054] 1. Vector digestion and fragment ligation

[0055] According to the reaction system of 1μl vector DNA (1μg / μl), 4μl 10*buffer, 32μl ddH2O, 1.5μl EcoRI, 1.5μl BamHI, react in a 37℃ water bath for 1-2h, digest pHBLV-U6-MCS-PGK-PURO, and perform agarose gel electrophoresis after digestion to recover the target fragment. TM The miR-92a-1-5p target fragment with sticky ends was connected to the pHBLV-U6-MCS-PGK-PURO vector after restriction digestion to obtain the miR-92a-1-5p overexpression recombinant lentiviral vector.

[0056] 2. Recombinant vector transformation

[0057] Thaw DH5α competent cells on ice, take 10 μl of the above-mentioned miR-92a-1-5p overexpression recombinant lentiviral vector and add it to 100 μl competent cells, tap the wall of the EP tube to mix, and let it stand on ice for 30 minutes; heat shock in a 42°C water bath for 45 seconds, and immediately cool it on ice for 2 minutes; add 900 μl of LB liquid culture medium without antibiotics to the EP tube, and shake the bacteria at 37°C 250rpm for 1 hour; centrifuge the transformed bacterial solution at 5,000rpm for 5 minutes, discard 900 μl of the supernatant and resuspend it with the remaining culture medium, and use a sterile coating rod to gently spread the resuspended bacterial solution on the LB solid culture medium plate with ampicillin preheated in a 37°C incubator in advance; incubate the plate coated with the bacterial solution inverted in a 37°C incubator for 12-16 hours.

[0058] 3. Colony PCR identification and sequencing

[0059] Pick 10 clones on the recombination reaction transformation plate and put them into 1.5ml EP tubes containing 1ml LB liquid medium containing ampicillin, shake the bacteria at 37℃250rpm for 12h, and pick a part of the bacterial solution for colony PCR identification. The amplification primers use the universal sequencing primers on the vector. The remaining bacterial solution corresponding to the colonies identified as positive by colony PCR is sequenced and sequenced (Shanghai Sangon Biotechnology Co., Ltd.) to select the successfully constructed target plasmid.

[0060] 4. Preparation of virus concentrate

[0061] 10 μg of miR-92a-1-5p overexpression recombinant lentiviral vector pHBLV-U6-MCS-PGK-PURO empty vector, 10 μg of virus packaging auxiliary plasmid pSPAX2 and 5 μg of pMD2G (Shanghai Hanheng Biotechnology Co., Ltd.) were transfected into 293T cells according to the instructions of Lipofectamine 3000 transfection reagent (Shanghai Hanheng Biotechnology Co., Ltd.). After 48 hours, the cell culture fluid was collected and centrifuged to obtain the viral supernatant, i.e., the miR-92a-1-5p overexpression / overexpression control virus concentrate, which was stored in a -80°C refrigerator.

[0062] 5. Cell infection

[0063] Add 20 μl of miR-92a-1-5p overexpression / overexpression control virus concentrate to human cardiac fibroblasts HCFs with a confluence rate of 70-80%; replace with fresh complete culture medium after 24 hours; use puromycin for positive screening and amplification culture after 48 hours to obtain human cardiac fibroblast cell line HCFs after virus infection screening.

[0064] 6. Obtaining cDNA and identifying the overexpression effect of miR-92a-1-5p by RT-qPCR

[0065] (1) RNA was extracted from human cardiac fibroblasts HCFs infected with miR-92a-1-5p overexpression virus concentrate and overexpression control virus concentrate using the RNA extraction kit provided by Shanghai Sangon Biotechnology Co., Ltd. Bulge-LoopTM miRNA RT Primer (20 μM) provided by Ruibo Biotechnology Co., Ltd. was added with an appropriate amount of RNase-free H2O to prepare Bulge-LoopTM miRNA RT Primer (5 μM). Finally, 10 μl of RT reaction system was mixed and centrifuged instantaneously. The reverse transcription reaction program was designed as follows: 42°C for 60 min, 70°C for 10 min, and finally cDNA was obtained by reverse transcription.

[0066] (2) RT-qPCR identification of miR-92a-1-5p overexpression effect

[0067] The RT-qPCR reaction system was prepared with a total volume of 20 μl, consisting of 10 μl of SYBR Green Mix (Ruibo Biotechnology Co., Ltd.) containing Taq enzyme, dNTP mix, PCR Buffer, SYBR Green I, etc., 0.6 μl of upstream primer, 0.6 μl of downstream primer, 8 μl of nuclease-free water, and 0.8 μl of cDNA template obtained in step 1. The prepared reaction system was placed on a fluorescent quantitative PCR instrument for RT-qPCR amplification reaction to detect the relative expression of miR-92a-1-5p in human microcardiac fibroblasts infected with miR-92a-1-5p overexpression virus (U6 gene was used as the internal reference gene). The relative expression of miR-92a-1-5p was calculated using the 2-ΔΔCT method.

[0068] The upstream primer for amplifying the miR-92a-1-5p gene was: 5'-AGGTTGGGATCGGTTGCAAT-3' (SEQ ID NO: 7), and the downstream primer was 5'-AGCATTGCAACCGATCCCAA-3' (SEQ ID NO: 8).

[0069] The upstream primer for amplifying the U6 gene was 5'-CTCGCTTCGGCAGCACA-3' (SEQ ID NO: 9), and the downstream primer was 5'-ACGCTTCACGAATTTGCGT-3' (SEQ ID NO: 10).

[0070] (3) Experimental results

[0071] Test results such as Figure 1 The results showed that after human cardiac fibroblasts were infected with miR-92a-1-5p overexpression lentiviral concentrate, the expression level of miR-92a-1-5p was significantly higher than that of the control, indicating that the overexpression effect of miR-92a-1-5p was significant.

[0072] Example 3: Effect of miR-92a-1-5p overexpression on the activation of human cardiac fibroblasts under TGF-β-induced fibrosis intervention conditions

[0073] 1. Modeling of human cardiac fibroblast activation induced by TGF-β recombinant protein

[0074] The miR-92a-1-5p overexpressing human cardiac fibroblast cell line and the control cell line constructed in Example 2 were seeded in a 6-well plate in advance, and 20 ng / mL of TGF-β recombinant protein was added after the cells adhered to the plate to induce fiber activation. The cells were treated under culture conditions of 5% CO2 and 37°C for 24 h.

[0075] 2. Western blot experiment:

[0076] Neutral lysis buffer RIPA was used to extract total cellular protein from human cardiac fibroblasts HCFs infected with miR-92a-1-5p overexpression virus concentrate and overexpression control virus concentrate. After denaturation, SDS-PAGE electrophoresis was performed at 100V for 90min; transfer to membrane at 300mA for 100min; PVDF membrane was blocked with rapid blocking buffer for 30min; β-actin rabbit primary antibody (Cell Signaling Technology, USA) was diluted 1000:1, Fibronectin rabbit primary antibody (Cell Signaling Technology, USA) was diluted 1000:1, FSP-1 rabbit primary antibody (Cell Signaling Technology, USA) was diluted 1000:1, and Vimentin rabbit primary antibody (Cell Signaling Technology, USA) was diluted 1000:1. Technology Company), 1000:1 diluted α-SMA rabbit primary antibody (Abcam, UK) and incubate at 4°C overnight; recover the primary antibody and wash the membrane 3 times with 1×TBST solution on a shaker, 10 min each time; incubate at room temperature for 1 h with 5000:1 diluted mouse secondary antibody (Cell Signaling Technology, USA); and develop.

[0077] 3. Experimental results

[0078] The results of Western blot development are as follows Figure 2 As shown. Under the induction of TGF-β, the expression levels of Fibronectin, Vimentin, α-SMA and FSP-1 in human cardiac fibroblasts increased significantly, indicating that TGF-β successfully induced fibroblast activation in human cardiac fibroblasts. Overexpression of miR-92a-1-5p significantly inhibited the expression levels of these proteins, indicating that miR-92a-1-5p can significantly improve the ability of human cardiac fibroblasts to activate fibers under TGF-β-induced fibrosis intervention conditions.

[0079] Example 4: Effect of miR-92a-1-5p overexpression on the inhibition of migration of human cardiac fibroblasts under TGF-β-induced fibrosis intervention conditions

[0080] 1. Transwell experiment

[0081] The human cardiac fibroblast cell line and control cell line overexpressing miR-92a-1-5p were digested with 0.25% trypsin, and centrifuged at 1000rpm for 5min after termination with complete medium; the cells were inoculated in the upper chamber of the Transwell chamber (Beijing Lanjieke Technology Co., Ltd.) with 20,000 cells per well and 200μl serum-free medium, and 500μl high-glucose DMEM medium containing 10% fetal bovine serum was added to the lower chamber of the transwell chamber, and TGF-β recombinant protein was added to the lower chamber to a final concentration of 20ng / μL to induce fiber activation. The culture plate was placed in an incubator at 37°C and 5% CO2 for 24h; fixed with 4% PFA at room temperature for 30 minutes, and stained with 0.1% crystal violet at room temperature for 10 minutes; image acquisition.

[0082] 2. Experimental results

[0083] Transwell migration and statistical analysis Figure 3 The results showed that compared with the control group, miR-92a-1-5p overexpression could significantly inhibit TGF-β-induced human cardiac fibroblast migration.

[0084] Example 5: Effect of miR-92a-1-5p overexpression on the inhibition of collagen contraction in human cardiac fibroblasts under TGF-β-induced fibrosis intervention conditions.

[0085] 1. Preparation of Cell Suspension

[0086] The human cardiac fibroblast cell line overexpressing miR-92a-1-5p and the control cell line were digested with 0.25% trypsin, and the cells were centrifuged at 1000 rpm for 5 min after termination with complete culture medium. The human cardiac fibroblast cells were resuspended in high-glucose DMEM culture medium to a density of 400,000 cells / mL, and the suspension was placed on ice for later use.

[0087] 2. Preparation of collagen containing fibroblasts

[0088] Add 12 μl of 0.1 mol / 1 NaOH and 23 μl of 10× DMEM medium containing phenol red to 200 μl of type I rat tail collagen (5 mg / ml) and mix immediately. Then add 760 μl of the above cell suspension placed on ice, mix the cell suspension and collagen thoroughly and add them to the 24-well plate immediately, 300 μl per well, to avoid the generation of bubbles. Place the well plate at 37°C for 1 hour to allow the gel to solidify, and add 500 μl of high-glucose DMEM medium containing 20 ng / μl TGF-β to each well to induce fibroblast activation and collagen contraction.

[0089] 3. Imaging and Analysis of Collagen Contraction

[0090] After 24 h of TGF-β-induced fiber activation and collagen contraction, the well plate was placed under a developer and imaged in white light mode to measure the collagen area and calculate the collagen contraction rate.

[0091] 4. Experimental Results

[0092] The results of the collagen contraction experiment are as follows Figure 4 The results showed that compared with the control group, miR-92a-1-5p overexpression could significantly inhibit TGF-β-induced collagen contraction in human cardiac fibroblasts.

[0093] The miR-92a-1-5p overexpressing human cardiac fibroblast cell line constructed by the present invention is a miR-92a-1-5p overexpressing stable cell line, which lays a foundation for in-depth research on the molecular mechanism of miR-92a-1-5p inhibiting fibrosis and the application of miR-92a-1-5p in the treatment of fibrotic diseases.

[0094] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A nucleotide fragment expressing miR-92a-1-5p, characterized in that: Its sequence is shown in SEQ ID NO:

4.

2. An expression vector, characterized in that: The expression vector comprises the target gene sequence shown in SEQ ID NO:

4.

3. The expression vector according to claim 2, characterized in that The expression vector is selected from a virus, fungus or bacteria expression vector.

4. A cell comprising the expression vector according to claim 2 or 3, characterized in that: The cells are human cardiac fibroblasts.

5. A method for preparing a recombinant lentiviral expression vector, characterized in that: include: The lentiviral vector pHBLV-U6-MCS-PGK-PURO was digested with restriction endonucleases EcoRI and BamHI, and the target sequence shown in SEQ ID NO: 4 was cloned into the lentiviral vector after the digestion to obtain a recombinant lentiviral vector.

6. A method for preparing a human cardiac fibroblast cell line with high expression of miR-92a-1-5p, characterized in that: The steps include: Step 1: The recombinant lentiviral vector is combined with the virus packaging auxiliary plasmids pSPAX2 and pMD2G to form a three-plasmid lentiviral system for virus packaging, and then transfected into 293T cells to obtain a virus liquid; the recombinant lentiviral vector is a recombinant lentiviral vector prepared by the preparation method according to claim 5; Step 2: Infect human cardiac fibroblast cell lines with the virus solution obtained in step 1, and screen and obtain fibroblast cell lines with high expression of miR-92a-1-5p, i.e., fibroblast cell lines with high expression of miR-92a-1-5p.

7. Use of the expression vector according to claim 2 or 3, or the recombinant lentiviral vector prepared by the preparation method according to claim 5 in any one of the following A1) to A10): A1) preparing a reagent for promoting the expression of miR-92a-1-5p; A2) preparing a product for improving fiber activation of human cardiac fibroblasts; A3) preparing a product for inhibiting the migration ability of human cardiac fibroblasts; A4) preparing a product for inhibiting collagen contraction in human cardiac fibroblasts; A5) preparing a product for constructing a cell model or animal model with high expression of miR-92a-1-5p; A6) promoting miR-92a-1-5p expression in vitro; A7) ability to improve fiber activation of human cardiac fibroblasts in vitro; A8) Inhibits the migration ability of human cardiac fibroblasts in vitro; A9) Inhibits the collagen contractility of human cardiac fibroblasts in vitro; A10) Construct a cell model with high expression of miR-92a-1-5p.

8. Use of the cell according to claim 4, or the human cardiac fibroblast cell line with high expression of miR-92a-1-5p prepared by the preparation method according to claim 5, in any one of the following B1) to B7): B1) preparing a product for improving fiber activation of human cardiac fibroblasts; B2) preparing a product for inhibiting the migration ability of human cardiac fibroblasts; B3) preparing a product for inhibiting collagen contraction in human cardiac fibroblasts; B4) preparing a product for constructing an animal model with high expression of miR-92a-1-5p; B5) ability to improve fiber activation of human cardiac fibroblasts in vitro; B6) Inhibit the migration ability of human cardiac fibroblasts in vitro; B7) Inhibits the collagen contractile ability of human cardiac fibroblasts in vitro. B8) Study the molecular mechanism by which miR-92a-1-5p inhibits cardiac fibrosis.

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