Application of miR-101-3p in preparation of medicine for treating myocardial fibrosis

Abstract

The invention discloses application of miR-101-3p in preparation of a medicine for treating myocardial fibrosis, and relates to the technical field of medicines. According to the invention, the influence of the miR-101-3p / RAC1 / PAK1 pathway on endothelial cells EndMT is verified by adopting human umbilical vein endothelial cells and utilizing molecular and cell biology means; and constructing an HFrMF mouse model, and verifying the feasibility of intervening the signal channel to alleviate MF. And a thought is provided for clarifying the pathological mechanism of heart failure and exploring a new treatment target.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the treatment of myocardial fibrosis, and more specifically to the application of miR-101-3p in the preparation of drugs for treating myocardial fibrosis. Background Technology

[0002] Heart failure (HF) is a leading cause of death and a growing global health problem. Despite significant progress in treatment, the 5-year mortality rate remains as high as 50%. Therefore, exploring effective treatments and targets to improve the survival rate and quality of life for HF patients is a pressing public health issue that needs to be addressed.

[0003] Myocardial fibrosis (MF) is a major cause of heart failure progression and an end-stage manifestation of many heart diseases. It is characterized by excessive proliferation of myofibroblasts, excessive collagen synthesis, and massive deposition of extracellular matrix, leading to decreased cardiac systolic and diastolic function, ultimately developing into heart failure (HF). Endothelial-to-mesenchymal transition (EndMT) is a specific type of epithelial-mesenchymal transition (EMT). It refers to endothelial cells acquiring a mesenchymal phenotype under specific physiological or pathological conditions and expressing typical markers of myofibroblast differentiation, such as α-SMA, vimentin, and collagen, while the expression of endothelial cell-specific markers (VE-cadherin, CD31) gradually disappears. Simultaneously, their function and biological characteristics undergo significant changes, resulting in strong survival and migration abilities. Studies have shown that EndMT is one of the main sources of myofibroblasts. During EndMT, endothelial cell phenotype changes, losing polarity and cell junctions, transforming into myofibroblasts, and promoting collagen deposition, thus leading to MF. This suggests that targeting and regulating the EndMT process of endothelial cells-myofibroblasts may be an effective way to alleviate MF, thereby achieving the goal of treating heart failure. However, its mechanism is complex and has not yet been fully elucidated, and new intervention targets still need to be identified. Recent studies have shown that RAC1 (Rac family small GTPase 1) may be an important regulatory target for organ fibrosis. However, no relevant studies have been conducted in HFrMF animal models, and whether RAC1 can serve as a therapeutic target for HF requires further verification.

[0004] microRNAs (miRNAs) are highly conserved non-coding single-stranded RNAs and important regulators of gene expression. They exert their effects by binding to target genes at the post-transcriptional level through complete or incomplete complementation, leading to gene degradation or inhibiting translation. In heart failure (HF), some miRNAs are also considered effective diagnostic biomarkers, such as miR-654-5p, miR-181c, and miR-212-3p. miRNAs are also considered effective regulators of end-mode mutation (EndMT); however, research on the role and mechanisms by which miRNAs alleviate heart failure by regulating EndMT is limited.

[0005] Therefore, how EndMT regulation affects MF and whether it is an effective disease target remains to be explored. Previous research in this application found that serum miR-101-3p levels were significantly downregulated in MF patients, while its target gene RAC1 was significantly upregulated in cardiac endothelial cells. Activated RAC1 has been shown to be beneficial in maintaining the mesenchymal state of cells. Therefore, based on this, this application proposes that miR-101-3p may target and inhibit the RAC1 / PAK pathway, preventing the EndMT process in endothelial cells and thus alleviating MF. Summary of the Invention

[0006] The purpose of this invention is to provide the application of miR-101-3p in the preparation of drugs for treating myocardial fibrosis. Using human umbilical vein endothelial cells (HUVECs), molecular and cell biological techniques are employed to verify the effects of the miR-101-3p / RAC1 / PAK1 pathway on endothelial cell endotoxemia (EndMT). Furthermore, an HFrMF mouse model is constructed to verify the feasibility of intervening in the above signaling pathways to alleviate myocardial fibrosis. This provides insights into elucidating the pathological mechanisms of heart failure and exploring new therapeutic targets.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides the application of miR-101-3p in the preparation of drugs for treating heart failure.

[0009] On the one hand, the present invention provides the application of miR-101-3p in screening drugs for the treatment of heart failure.

[0010] On the one hand, the present invention provides the application of substances that promote miR-101-3p overexpression in the preparation of drugs for treating heart failure.

[0011] Preferably, the heart failure is heart failure caused by myocardial fibrosis.

[0012] Preferably, the myocardial fibrosis is myocardial fibrosis caused by endothelial-mesenchymal transition.

[0013] Preferably, the drug is a miR-101-3p agonist or a substance that promotes miR-101-3p overexpression.

[0014] Preferably, the target of miR-101-3p is RAC1.

[0015] Preferably, the target pathway of miR-101-3p is the RAC1 / PAK pathway.

[0016] On one hand, the present invention provides a medicament for treating myocardial fibrosis, the medicament comprising a substance that promotes the overexpression of miR-101-3p.

[0017] Preferably, the drug comprises an agonist of miR-101-3p.

[0018] Preferably, it also includes pharmaceutically acceptable excipients.

[0019] Preferably, the formulation of the drug is an injection or an oral preparation, wherein the oral preparation is a solid oral preparation or a liquid oral preparation, wherein the solid oral preparation is a capsule, tablet, powder, granule or pill, and the liquid oral preparation is a suspension or solution.

[0020] On one hand, the present invention provides a method for screening candidate drugs for treating heart failure, comprising the following steps:

[0021] (1) Treat systems that express or contain miR-101-3p with the analyte;

[0022] (2) Detect the expression level of miR-101-3p in the system described in step (1);

[0023] (3) Select the test substance that promotes the expression level of miR-101-3p as a candidate drug.

[0024] Compared with existing technologies, the beneficial effects of this solution are:

[0025] This invention, through cell and animal experiments, revealed that miR-101-3p is significantly downregulated in patients with myocardial fibrosis, clarifying that miR-101-3p is its target. The regulation of EndMT and vascular endothelial function by miR-101-3p in HUVECs depends on the RAC1 signaling pathway. Overexpression of miR-101-3p and inhibition of RAC1 can reverse EndMT protein expression. This provides a new approach for screening or preparing drugs for heart failure. Attached Figure Description

[0026] Figure 1Screening for differentially expressed miRNAs (AB are volcano plots and heatmaps of differential expression of miRNAs in cohort GSE104150; CD are volcano plots and heatmaps of differential expression of miRNAs in cohort GSE196421; E is the intersection of differential expression of miRNAs in cohorts GSE104150 and GSE196421).

[0027] Figure 2 The expression of five miRNAs in the myocardial fibrosis-related cohorts is shown in A, C, E, G, and I (expression of five miRNAs in cohort GSE140150, expression of five miRNAs in cohort GSE96421, and expression of five miRNAs in cohort GSE96421). The five miRNAs are hsa-miR-15a-5p, hsa-miR-101-3p, hsa-miR-144-3p, hsa-miR-190a-5p, and hsa-miR-494-3p, respectively.

[0028] Figure 3 Prediction and functional enrichment analysis of hsa-miR-101-3p target genes (A: intersection of hsa-miR-101-3p target genes predicted by the mirtarbase and rnainter databases; B: functional enrichment analysis of target genes; C: PPI network analysis).

[0029] Figure 4 The expression of the hsa-miR-101-3p target gene RAC1 in cardiac cell populations;

[0030] Figure 5 The cell morphology of HUVECs induced by different concentrations of TGFb for 48 h under low-density and high-density conditions (Con represents the blank control group).

[0031] Figure 6 The image shows the cell morphology after miR-101-3p mimic / inhibitor transfection (mimic represents miR-101-3p overexpression transfection group, mimic NC represents the negative control group of miR-101-3p overexpression; inhibitor represents miR-101-3p knockdown transfection group, inhibitor NC represents the negative control group of miR-101-3p knockdown).

[0032] Figure 7 Comparison of miR-101-3p expression levels after 48 hours of induction with different concentrations of TGFb;

[0033] Figure 8Comparison of intracellular miR-101-3p expression after miR-101-3p mimic / inhibitor transfection (A: intracellular miR-101-3p expression after miR-101-3p mimic transfection, B: intracellular miR-101-3p expression after miR-101-3p inhibitor transfection).

[0034] Figure 9 The effects of miR-101-3p overexpression and knockdown on related proteins in HUVEC (A and E are immunoblot images of related proteins; B, C, D, F, G, and H are statistical comparisons of the relative content of RAC1, PAK1, αSMA, Vimentin, VEcad, and CD31 proteins, respectively).

[0035] Figure 10 Results of cell scratch assay after transfection with miR-101-3p mimic / inhibitor;

[0036] Figure 11 Comparison of the angiogenic ability of HUVEC cells after transfection with miR-101-3p mimic / inhibitor;

[0037] Figure 12 Comparison of relative luciferase activity between miR-101-3p mimic and RAC1-WT wild-type and RAC1-MT mutant co-transfected groups (A is the vector map of pmirGLO plasmid and the results of miRNA target gene verification; B is the comparative analysis of relative luciferase activity).

[0038] Figure 13 The effects of RAC1 overexpression and knockout on related proteins are shown in Figure A (Immunoblot images of related proteins; B, C, D, E, F, and G are statistical comparisons of the relative content of RAC1, PAK1, SMA, Vimentin, VEcad, and CD31 proteins, respectively; where sh-RAC1 represents the RAC1 knockout transfection group, sh-NC is the negative control, RAC1 represents the RAC1 overexpression transfection group, and PC-NC is the positive control).

[0039] Figure 14 The expression analysis of related proteins after RAC1 overexpression and co-transfection with miR-101-3p mimic was performed (A is the immunoblot image of related proteins; B, C, D, E, F, and G are the statistical comparison of the relative content of RAC1, PAK1, SMA, Vimentin, VEcad, and CD31 proteins, respectively; among them, mimic+RAC1 is the miR-101-3p mimic+RAC1 overexpression co-transfection group).

[0040] Figure 15 Expression analysis of related proteins after RAC1 knockdown and co-transfection with miR-101-3p inhibitor (A is the immunoblot image of related proteins; B, C, D, E, F, and G are the statistical comparison of the relative content of RAC1, PAK1, SMA, Vimentin, VEcad, and CD31 proteins, respectively. Among them, inhibitor+sh-RAC1 or inhibitor+si-RAC is the group co-transfected with miR-101-3p inhibitor and RAC1 overexpression vector).

[0041] Figure 16 HE staining pathological features of cardiomyocytes in a mouse model of myocardial fibrosis with miR-101-3p overexpression and RAC1 inhibition (Ctrl represents blank control, Model represents model group, miR-101-3p Ago represents miR-101-3p overexpression, Ago NC represents negative control, RAC1 inhibitor represents RAC1 inhibition, and Combined represents the combination of miR-101-3p overexpression and RAC1 inhibition).

[0042] Figure 17 WGA staining pathological features of cardiomyocytes in HFrMF mouse model of miR-101-3p overexpression and RAC1 inhibition;

[0043] Figure 18 Electron microscopy findings of cardiomyocytes in an HFrMF mouse model of miR-101-3p overexpression and RAC1 inhibition.

[0044] Figure 19 Immunofluorescence characteristics of cardiomyocytes in HFrMF mouse model of miR-101-3p overexpression and RAC1 inhibition;

[0045] Figure 20 The expression of cardiomyocyte-related proteins in the HFrMF mouse model was compared between miR-101-3p overexpression and RAC1 inhibition (A is the immunoblot image of related proteins; B, C, D, E, F, and G are the statistical comparison of the relative content of RAC1, PAK1, Vimentin, VEcad, SMA, and CD31 proteins, respectively; where P represents the metoprolol group). Detailed Implementation

[0046] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0050] Example 1: Effect of miRNA expression on HFrMF progression

[0051] This embodiment analyzed miRNA expression information from the GEO database using bioinformatics technology, and found five common differentially expressed miRNAs in the GSE104150 and GSE196421 cohorts: hsa-miR-15a-5p, hsa-miR-101-3p, hsa-miR-144-3p, hsa-miR-190-5p, and hsa-miR-494-3p (see [link]). Figure 1 Excluding hsa-miR-494-3p, which showed the opposite expression trend, the p-value revealed a highly significant difference in hsa-miR-101-3p expression between patients with myocardial fibrosis and healthy individuals (see [link to article]). Figure 2 Therefore, target genes of hsa-miR-101-3p were predicted using Raninter and Mirtarbase, respectively. The results showed that 5755 target genes were predicted in the Raninter database and 347 target genes were predicted in the Mirtarbase database. Taking the intersection of the top 200 target genes with the highest confidence levels predicted from each database yielded 70 overlapping target genes (see...). Figure 3(A) Enrichment analysis of these 70 target genes revealed that they were mainly enriched in processes such as Regulation of cellular response to stress, EED-EZH2 complex, TGF-β signaling pathway, and Regulation of mitotic cell cycle (see A). Figure 3 (B in the text). Subsequent PPI network analysis revealed that six genes—RAC1, APP, CDKN1A, FOS, ZEB1, and TGFBR3—were enriched in the TGF-β signaling pathway (see [link to PPI analysis]). Figure 3 (C in the text). The HPA database confirmed that RAC1 is expressed in various cardiac tissue cells, with the highest abundance in endothelial cells among the top 10 genes with the highest confidence levels (see [link to HPA database]). Figure 4 Current research indicates that RAC1 can promote the epithelial-mesenchymal transition (EMT) of tumor cells, which is related to PAK1 pathway activation, suggesting that miR-101-3p has a potential role in regulating EMT. Furthermore, hsa-miR-101-3p was significantly downregulated in heart failure patients in the GSE104150 and GSE196421 cohorts.

[0052] In summary, downregulation of hsa-miR-101-3p can affect the mesenchymal transition process by regulating the expression of the endothelial cell target gene RAC1.

[0053] Example 2: Effect of miR-101-3p on phenotypic transformation of HUVECs mesenchymal cells

[0054] To investigate the effect of miR-101-3p on the mesenchymal phenotype transformation of HUVECs, this study induced HUVEC cells with TGF-β. HUVEC cells were revived, passaged, and seeded in six-well plates, and induced for 48 hours with different concentrations of TGF-β (0, 10 ng / ml, 20 ng / ml). Cell morphology changes were observed under a microscope. The cell number per well was 15 weeks at low concentration and 30 weeks at high concentration. The results showed that HUVECs induced with 10-20 ng / ml TGF-β for 48 hours under both low and high density conditions underwent a gradual transformation from an endothelial cell cobblestone shape to a mesenchymal spindle shape, suggesting that TGF-β induces the mesenchymal transformation of HUVECs (see [link to study].) Figure 5 Subsequently, RNA was extracted from HUVEC cells after induction treatment, and miR-101-3p / U6 was detected by RT-qPCR. The results showed that miR-101-3p expression was significantly reduced 48 h after TGFb induction, suggesting that TGFb induces a decrease in miR-101-3p (see...). Figure 7 ).

[0055] This embodiment also included a HUVEC cell transfection experiment. One day before transfection, cells were counted, and 1 ml was added to each well of a 6-well plate to achieve a cell density of 150,000 cells. For each well, 5 μL of siRNA was diluted with 250 μL of serum-free medium to a final concentration of 100 nM, and incubated at room temperature for 5 min. Experimental and control groups were set up: the experimental group contained miR-101-3pinhibitor and miR-101-3p mimic, and the control group contained inhibitor NC and mimic NC. For each well, 10 μL of Lipo3000 (ThermoFisher) was diluted with 250 μL of serum-free medium and incubated at room temperature for 5 min. The two incubation solutions were then mixed, and incubation continued at room temperature for 20 min. Adhering cells were then replaced with the aforementioned mixed serum-free complex and cultured at 37°C in 5% CO2 for 6 hours, followed by culture in serum-added growth medium at 37°C in 5% CO2 for 48 hours. Cell samples were collected, washed twice with PBS, the liquid was discarded, cell morphology was recorded, and then RT-qPCR was performed. After transfecting cells were induced with 10 ng / ml TGFb for 48 h, whole protein was extracted and Western blot was performed.

[0056] Cell morphology results showed that the miR-101-3p mimic transfection group exhibited a more pronounced cobblestone morphology, typical of HUVECs, compared to the control group (mimic NC). Conversely, the miR-101-3p inhibitor knockdown group showed a more spindle-shaped morphology compared to the negative control. This suggests that inhibiting miR-101-3p exacerbates the mesenchymal transition (see...). Figure 6 RT-qPCR results showed that transfection with mimic significantly increased intracellular miR-101-3p expression. Conversely, transfection with miR-101-3p inhibitor significantly knocked down intracellular miR-101-3p expression, indicating that both miR-101-3p mimic and miR-101-3p inhibitor transfections were successful (see [link to RT-qPCR results]). Figure 8 Western blot analysis showed that miR-101-3p overexpression inhibited EndMT marker proteins αSMA and Vimentin in HUVECs, while promoting endothelial cell marker proteins VEcad and CD31. This suggests that miR-101-3p regulates the expression of EndMT and endothelial marker proteins (see...). Figure 9 ).

[0057] Example 3: Effects of miR-101-3p on mesenchymal cell function in HUVECs

[0058] To investigate the effects of miR-101-3p overexpression and knockdown on HUVECs mesenchymal cell function, this example performed cell scratch assays and in vitro vascular mimicry assays on transfected HUVECs.

[0059] Cell scratch assay: HUVEC cells transfected for 48 hours in Example 2 were digested and seeded evenly into 6-well plates (800,000 cells per well). After complete cell adhesion, scratches were created using a 200 μL sterile pipette tip. The cells were gently washed twice with PBS and then replaced with basal medium. Microscopic images were taken at 0 h and 24 h. 4x magnification was preferred. ImageJ was used to analyze the relative change in scratch area. The relative migration rate was calculated in Excel as: Migration rate = (Area(24h) - Area(0)) / Area(0).

[0060] The results showed that the migration rate was significantly increased in the miR-101-3p overexpression group. This suggests that miR-101-3p can increase migration rate (see...). Figure 10 ).

[0061] In vitro vascular mimicry assay: The day before the experiment, Matrigel was placed in an icebox at 4°C, and pre-chilled pipette tips and 24-well plates were prepared. 100 μL of Matrigel (diluted 1:2 with pre-chilled PBS) was added to each well, avoiding air bubbles, and quickly rotated to spread evenly. The plates were placed in a humidified chamber and incubated for approximately 1 hour to allow the gel to solidify. HUVEC cells in the logarithmic growth phase were digested, counted, and 30,000 cells / well were aspirated. The cells were centrifuged at 2000 rpm for 3 minutes to obtain the cell pellet. The cells were resuspended in 500 μL of the collected supernatant (with 10 μL of FBS added) and seeded into the prepared Matrigel-coated plates. Observation and photography were performed after 24 hours.

[0062] The results showed that HUVEC cells overexpressing miR-101-3p exhibited enhanced angiogenesis, while those knocked down showed diminished angiogenesis, suggesting that miR-101-3p increases the angiogenesis capacity of HUVEC cells (see [link to study]. Figure 11 ).

[0063] Example 4: Validation experiment of miR-101-3p targeting and binding to RAC1

[0064] To verify the targeting relationship between miR-101-3p and the target gene RAC1 in HUVEC cells, this example performed a dual-luciferase activity assay, including the following steps:

[0065] (1) Construction of dual-luciferase gene reporter plasmid:

[0066] 1) Locate the 3'UTR sequence of RAC1 on NCBI, predict the binding site of miR-101-3p (MIMAT0000099) to the target gene RAC1 using Targetscan (TargetScanHuman 8.0), and perform site-directed mutagenesis on several bases in the miR-101-3p binding site region. The information is shown in the table below:

[0067]

[0068] 2) Preparation of wild-type (WT) plasmids and mutant (MT) plasmids: pmirGLO WT / MT plasmids with miR-101-3p targeting RAC1 binding sites were synthesized by Suzhou Hongxun and endotoxin-free plasmids were extracted.

[0069] (2) Dual-luciferase reporter gene assay:

[0070] 1) Cell transfection: The experimental groups were set up as follows: miR-101-3p mimics+RAC1-UTR-WT group (denoted as mimics+RAC1-WT), NC Mimics+RAC1-UTR-WT group (denoted as NC-WT), miR-101-3p mimics+RAC1-UTR-MT group (denoted as mimics+RAC1-MT), and NC Mimics+RAC1-UTR-MT (denoted as NC-MT).

[0071] 2) After 48 hours, remove the cells, discard the old culture medium, and wash three times with pre-cooled PBS.

[0072] 3) The ratio of the intensity of firefly luciferin (RLU) to that of sea urchin fluorescence (RLU) was calculated using a dual-luciferase reporter gene assay kit (Cat#:RG009, Beyotime), and the reporter gene activity was detected.

[0073] The results showed that the relative luciferase activity was significantly reduced in the co-transfection group with miR-101-3p overexpression and RAC1-WT wild-type, while there was no significant difference between the co-transfection group with RAC1-MT mutant and miR-101-3p overexpression. This suggests that miR-101-3p binds to the RAC1-WT gene (see...). Figure 12 ).

[0074] Example 5: Effects of RAC1 on EndMT and Vascular Mimicry in HUVECs

[0075] In this embodiment, RAC1 silencing vector (sh-RAC1), RAC1 overexpression vector (RAC1), miR-101-3p mimic+RAC1 overexpression vector (mimic+RAC1), and miR-101-3p inhibitor+RAC1 overexpression vector (inhibitor+sh-RAC1 or inhibitor+si-RAC) were transfected into HUVECs cells. Cells were treated with 10 ng / ml TGFb for 48 h after transfection. The complete protein was then extracted and analyzed by Western blotting.

[0076] The results showed that RAC1 overexpression upregulated PAK1 and EndMT proteins SMA and Vimentin, while downregulating endothelial cell markers related to vascular homeostasis, VE-cad and CD31, suggesting the effect of RAC1 overexpression / knockdown on EndMT in HUVECs (see [link to study]). Figure 13 RAC1 overexpression counteracts the effects of miR-101-3p on the regulation of PAK1, EndMT proteins SMA and Vimentin, and endothelial cell markers related to vascular homeostasis such as VE-cad and CD31, suggesting that RAC1 overexpression may negate the effects of miR-101-3p (see [link to article]). Figure 14 RAC1 knockdown counteracts the regulation of PAK1, EndMT proteins SMA and Vimentin, and endothelial cell markers related to vascular homeostasis such as VE-cad and CD31 by miR-101-3p inhibitor. This suggests that RAC1 knockdown negates the effects of miR-101-3p inhibitor (see...). Figure 15 ).

[0077] The above results indicate that miR-101-3p regulates EndMT and vascular endothelial function in HUVECs in response to the RAC1 signaling pathway.

[0078] Example 6 Animal Experiment

[0079] In this embodiment, a mouse model of myocardial fibrosis (HFrMF) was established by ligation of the anterior descending artery. After successful model establishment, mice were randomly divided into the model group (Model), metoprolol group (P), miR-101-3pagomir group (miR-101-3pAgo), miR-101-3pagomir NC group (Ago NC), RAC1 inhibitor group (RAC1 inhibitor), and miR-101-3pagomir + RAC1 inhibitor group (Combined). The blank group (Ctrl) and the sham operation group (Model) served as controls. After two weeks of continuous intervention, the cardiac function indicators of each group of mice were detected by small animal echocardiography. After the echocardiography examination, the mice were sacrificed, and blood and hearts were collected. HE staining and WGA staining were used to observe the pathological changes of myocardial tissue in each group of mice; transmission electron microscopy was used to observe the changes in the ultrastructure of endothelial cells; immunofluorescence was used to examine the expression of EndMT markers (α-SMA, vimentin, CD31, VE-Cadherin); and Western blotting was used to examine the expression of the RAC1 / PAK1 signaling pathway.

[0080] HE staining results showed that cardiomyocytes were enlarged and fibrosis was significantly increased in the Model group. miR-101-3p Ago and inhibition of RAC1 could reverse these cardiomyocyte changes. This suggests that miR-101-3p Ago and inhibition of RAC1 can alleviate myocardial fibrosis (see...). Figure 16 WGA staining results showed that cardiomyocytes in the Model group were enlarged, and miR-101-3p Ago and inhibition of RAC1 could reverse the cardiomyocyte enlargement. This suggests that miR-101-3p Ago and inhibition of RAC1 can reverse cardiomyocyte enlargement (see...). Figure 17 Electron microscopy results showed that the model group exhibited myocardial fiber rupture and disordered arrangement, as well as mitochondrial swelling and deformation. The drug intervention group showed better myocardial fiber structural integrity than the model group. Specifically, the miR-101-3p-Ago and combined groups showed myocardial fiber structure similar to the Ctrl group, with mitochondrial swelling alleviated compared to the model group. This suggests that miR-101-3p-Ago and RAC1 inhibition can reverse myocardial fiber swelling (see...). Figure 18 Immunofluorescence results showed that miR-101-3p Ago and inhibition of RAC1 reduced the expression of EndMT-related proteins in Model. This suggests that miR-101-3p Ago and inhibition of RAC1 can reverse EndMT protein expression (see...). Figure 19Western blot results showed that RAC1 / PAK1 protein expression was increased in the Model group, along with increased expression of EndMT marker proteins SMA and Vimentin, and decreased expression of endothelial cell marker proteins VE-cad and CD31. miR-101-3p Ago and RAC1 inhibition reduced the expression of EndMT-related proteins in the Model. This suggests that miR-101-3p Ago and RAC1 inhibition can reverse EndMT protein expression (see...). Figure 20 ).

[0081] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

[0082] Based on the above embodiments, the present invention also provides a method for screening candidate drugs for treating heart failure, comprising the following steps: (1) treating a system expressing or containing miR-101-3p with a test substance; (2) detecting the expression level of miR-101-3p in the system of step (1); and (3) selecting a test substance that can promote the expression level of miR-101-3p as a candidate drug.

[0083] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. Application of miR-101-3p in the preparation of drugs for treating heart failure.

2. Application of miR-101-3p in screening drugs for the treatment of heart failure.

3. Application of substances that promote miR-101-3p overexpression in the preparation of drugs for treating heart failure.

4. The application according to any one of claims 1-3, characterized in that, The heart failure mentioned refers to heart failure caused by myocardial fibrosis.

5. The application according to claim 4, characterized in that, The myocardial fibrosis mentioned is myocardial fibrosis caused by endothelial-mesenchymal transition.

6. The application according to claim 4, characterized in that, The drug is an agonist of miR-101-3p or a substance that promotes the overexpression of miR-101-3p.

7. The application according to claim 4, characterized in that, The target of miR-101-3p is RAC1.

8. The application according to claim 4, characterized in that, The target pathway of miR-101-3p is the RAC1 / PAK pathway.

9. A drug for treating myocardial fibrosis, characterized in that, The drug includes substances that promote the overexpression of miR-101-3p.

10. The medicament according to claim 8, characterized in that, The drug includes an agonist of miR-101-3p.

11. The medicament according to claim 8 or 9, characterized in that, It also includes pharmaceutically acceptable excipients.

12. The medicament according to claim 10, characterized in that, The drug is formulated as an injection or an oral preparation, wherein the oral preparation is a solid oral preparation or a liquid oral preparation, wherein the solid oral preparation is a capsule, tablet, powder, granule or pill, and the liquid oral preparation is a suspension or solution.

13. A method for screening candidate drugs for the treatment of heart failure, characterized in that, Includes the following steps: (1) Treat systems that express or contain miR-101-3p with the analyte; (2) Detect the expression level of miR-101-3p in the system described in step (1); (3) Select the test substance that promotes the expression level of miR-101-3p as a candidate drug.

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