A computer-assisted drug screening method, system and device based on CST7 and miRNA

Abstract

The present invention discloses a computer-assisted drug screening method, system and device based on CST7 and miRNA, which mainly include the CST7 gene, hsa-miR-18a-3p and / or hsa-miR-3130-5p. The computer-assisted drug screening method, system and device of the present invention are used to screen compounds that regulate the binding ability between CST7 and miRNA. The present invention also provides the use of CST7 and miRNA in preparing a pharmaceutical composition for regulating myocardial hypertrophy disease and the use of CST7 and miRNA in screening candidate drugs for regulating myocardial hypertrophy disease.

Description

Technical Field

[0001] The present invention belongs to the field of computer bioinformatics, and specifically relates to a computer-assisted drug screening method, system and device based on CST7 and miRNA. Background Art

[0002] Hypertrophic cardiomyopathy (HCM) is a myocardial disease of unknown etiology, characterized by asymmetric thickening of the ventricular wall, often invading the ventricular septum. This leads to a reduction in the ventricular cavity, impaired left ventricular filling, and decreased left ventricular diastolic compliance. HCM is categorized as either obstructive or non-obstructive depending on the presence or absence of left ventricular outflow tract obstruction, and may be related to genetic factors. HCM carries a risk of sudden death and is a cause of sudden death during exercise.

[0003] The cystatin superfamily comprises proteins containing multiple cystatin-like sequences. Some members are active inhibitors of cysteine ​​proteases, while others have lost or may never have acquired this inhibitory activity. Three inhibitory families exist within this superfamily, including type 1 cystatins (steins), type 2 cystatins, and kininogens. Type 2 cystatin proteins are a class of cysteine ​​protease inhibitors found in various human fluids and secretions. The CST7 gene encodes a glycosylated cysteine ​​protease inhibitor that is postulated to play a role in immunomodulation by inhibiting a unique target in the hematopoietic system. Expression of this protein has been observed in various human cancer cell lines established from malignant tumors. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] The present invention provides a computer-assisted drug screening method based on CST7 and miRNA, the method specifically comprising:

[0006] Obtain CST7 gene data and miRNA gene data;

[0007] The sequence information and spatial structure of the CST7 gene and the sequence information and spatial structure of the miRNA gene are selected to determine the binding sites of the two as the binding sites of the targeted drug;

[0008] A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

[0009] Furthermore, the computer-assisted drug screening method process is as follows:

[0010] Obtain the binding sites of CST7 gene and miRNA gene;

[0011] Based on the sequence information and spatial structure of the binding sites between the CST7 gene and the miRNA gene, compounds with similar sequences and structures were screened in the molecular database;

[0012] Molecular docking is performed between the screened compounds and the CST7 gene to calculate the binding energy scores of the targeted compounds, and candidate drugs are obtained by ranking according to the scores.

[0013] Furthermore, the computer-assisted drug screening method process is as follows:

[0014] Obtain the binding sites of CST7 gene and miRNA gene and the spatial structure of the complex after binding;

[0015] Based on the binding sites of CST7 gene and miRNA gene and the spatial structure of the complex after binding, compounds with complementary structures were screened in the molecular database;

[0016] Molecular docking is performed on the screened compounds and the complex formed by binding of the CST7 gene and the miRNA gene, the binding energy scores of the targeted compounds are calculated, and candidate drugs are obtained by ranking according to the scores.

[0017] Furthermore, the miRNA gene is hsa-miR-18a-3p and / or hsa-miR-3130-5p.

[0018] Furthermore, the compound includes protein analogs, antibodies, DNA, and RNA.

[0019] Furthermore, the compound includes various pharmaceutically acceptable salt forms.

[0020] Furthermore, the DNA includes single-stranded DNA, closed-circular DNA, and linked DNA.

[0021] Furthermore, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, scRNA, RNA with catalytic activity, and various viral RNAs.

[0022] Furthermore, the DNA includes DNA of various conformations, specifically including A-type, B-type, C-type, D-type, E-type, H-type, L-type, P-type, and Z-type.

[0023] Furthermore, the antibody includes dAb, Fab, Fab', scFv, Fv, disulfide-bonded Fv, or contains a single immunoglobulin variable domain.

[0024] Furthermore, the protein analogs include proteins artificially synthesized through protein engineering.

[0025] In some embodiments, the computer-assisted drug screening screens candidate drugs based on the binding site of the CST7 gene and the miRNA gene or the spatial conformation of the complex of the CST7 gene and the miRNA gene.

[0026] In some embodiments, the compound includes, but is not limited to, oxides, acids, bases, salts, and organic matter. In some embodiments, the oxide is a compound composed of two elements, one of which is oxygen. In some embodiments, the acid is a compound in which all cations produced upon ionization are hydrogen ions. In some embodiments, the base is a compound in which all anions produced upon ionization are hydroxide ions. In some embodiments, the salt is a compound that produces metal cations (or ammonium ions) and acid ions upon ionization. In some embodiments, the organic matter is a general term for carbon-containing compounds (excluding carbon monoxide, carbon dioxide, carbonates, metal carbides, and cyanides) or hydrocarbons and their derivatives.

[0027] In some embodiments, pharmaceutically acceptable salt forms refer to pharmaceutically acceptable salts, including salts prepared from pharmaceutically acceptable bases or acids.

[0028] In some embodiments, examples of pharmaceutically acceptable bases include, but are not limited to, sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, magnesium, aluminum, primary amines, secondary amines, tertiary amines, substituted amines (including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins) such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins.

[0029] In some embodiments, examples of pharmaceutically acceptable acids include, but are not limited to, inorganic and organic acids, such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, vinylsulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid.

[0030] The present invention provides a computer-assisted drug screening system based on CST7 and miRNA, comprising:

[0031] Data acquisition unit: obtain CST7 gene data and miRNA gene data;

[0032] Data processing unit: selects the sequence information and spatial structure of the CST7 gene and the sequence information and spatial structure of the miRNA gene, and determines the binding sites of the two as the binding sites of the targeted drug;

[0033] Drug screening unit: A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site. The computer-assisted drug screening method is the computer-assisted drug screening method among the computer-assisted drug screening methods described above.

[0034] In some embodiments, the units include elements (such as software elements, object-oriented software elements, class elements, and task elements), processors, functions, properties, procedures, subroutines, program code segments, drivers, firmware, microcode, circuits, data, databases, data structures, tables, and arrays.

[0035] The present invention provides the use of CST7 and miRNA in any of the following:

[0036] 1) Application in the preparation of pharmaceutical compositions for regulating myocardial hypertrophy;

[0037] 2) Application in screening candidate drugs for regulating myocardial hypertrophy;

[0038] Wherein, the miRNA gene is hsa-miR-18a-3p and / or hsa-miR-3130-5p.

[0039] The present invention provides a computer-assisted drug screening device based on CST7 and miRNA, comprising:

[0040] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the computer-assisted drug screening method described above is implemented.

[0041] The present invention provides a computer-readable storage medium having a computer program thereon, comprising:

[0042] When the computer program is executed by a processor, the computer-assisted drug screening method described above is implemented.

[0043] The present invention provides a drug, which is obtained by screening using the computer-assisted drug screening method described above.

[0044] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0045] Furthermore, the auxiliary materials include one or more of diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

[0046] The term "unit" as used in the present invention refers to an application specific integrated circuit (ASIC), an electronic circuit, a processor (such as a shared processor, a dedicated processor or a group processor, etc.) and memory for executing one or more software or firmware programs, a combined logic circuit and / or other suitable components that support the described functions.

[0047] The term "device" as used in the present invention is not limited to one or a specific number of physical objects.

[0048] The term "processor" as used herein refers to any type of processor, including more than one processor, such as a multi-core design or multiple processors each having a multi-core design. The processor can be configured to execute a sequence of computer program instructions, such as a sequence of those instructions stored in a memory, to perform various operations, processes, and methods according to the exemplary embodiments of the present invention.

[0049] The term "memory" as used herein refers to any type of long-term, short-term, volatile, nonvolatile or other memory and should not be limited to any particular type of memory or any particular number of memories or types of media storing memory. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The figure is a flowchart of a computer-assisted drug screening method based on CST7 and miRNA provided by an embodiment of the present invention.

[0051] Figure 2 Schematic diagram of a computer-assisted drug screening system based on CST7 and miRNA provided by an embodiment of the present invention.

[0052] Figure 3 Schematic diagram of a computer-assisted drug screening device based on CST7 and miRNA provided by an embodiment of the present invention.

[0053] Figure 4 Figure 1 is the result of screening candidate genes for HCM-related macrophages, where A is the differentially expressed gene analysis of our own sequencing dataset, B is the differentially expressed gene analysis of the GSE141910 dataset, C is the overlap analysis of differentially expressed genes in the two datasets, D is the intersection analysis of differentially expressed genes and macrophage-related genes, and E is the expression level of key candidate genes in the HCM group and the control group. HCM group n=8, control group n=8, data are expressed as Mean±SD, n=6, * indicates P<0.05, indicates P<0.01, and * indicates P<0.001.

[0054] Figure 5Figure 1 shows a significant causal association between the CST7 gene and the development of HCM. Figure A shows the Mendelian randomization analysis of the CST7 gene and HCM. nSNP represents the number of single nucleotide polymorphisms analyzed, and the p-value represents the level of statistical significance. Figure B shows a scatter plot of the relationship between the CST7 gene and HCM and the fitting results of different MR algorithms. The horizontal axis represents the effect of the SNP on CST7 gene expression (exposure), and the vertical axis represents the effect of the SNP on HCM risk (outcome). Colored lines represent the fitting results of different Mendelian randomization algorithms (such as IVW, MREgger, and weighted median). A positive slope indicates that the CST7 gene is a risk factor for HCM, while a negative slope indicates that it is a protective factor. Figure C shows a forest plot of the relationship between the CST7 gene and HCM. Figure D shows a funnel plot of the relationship between the CST7 gene and HCM. Figure E shows a sensitivity analysis of the relationship between the CST7 gene and HCM using Mendelian randomization.

[0055] Figure 6 Figure 1 shows that AAV-mediated macrophage-targeted CST7 gene overexpression significantly improved cardiac pathology in mice. Figure A shows CST7 mRNA expression analysis. RT-qPCR was used to assess CST7 mRNA expression in cardiac tissues of mice in each group. B. HE staining assessed cardiac structural changes. C. WGA staining assessed cardiomyocyte hypertrophy. D. Masson staining assessed collagen fiber deposition. E. Macroscopic observation of cardiac size in each group. F. Electrocardiographic voltage changes. G. Interventricular septal thickness (IVSs) measured by cardiac ultrasound. N = 10 for the HCM group and 10 for the control group. Data are expressed as mean ± SD, n = 6. * indicates P < 0.05, * indicates P < 0.01, and * indicates P < 0.001.

[0056] Figure 7Figure 1 shows the differential expression of miRNAs and circRNAs in HCM patients and healthy controls, along with the CST7 regulatory network. Figure A shows a volcano plot of differentially expressed miRNAs. Red dots indicate upregulated miRNAs, blue dots indicate downregulated miRNAs, and gray dots indicate miRNAs with no significant differences. The top 10 miRNAs with the most significant up- and downregulated differences are labeled. Figure B shows a heat map of differentially expressed miRNAs. The upper portion shows a heat map of the expression density of the top 10 upregulated and downregulated miRNAs in the samples, displaying five quantiles and the mean line. The lower portion shows a heat map of the expression of the top 10 upregulated and downregulated miRNAs in the samples. Figure C shows a volcano plot of differentially expressed circRNAs. Red dots indicate upregulated circRNAs, blue dots indicate downregulated circRNAs, and gray dots indicate circRNAs with no significant differences. The top 10 circRNAs with the most significant up- and downregulated differences are labeled. D shows a heatmap of differentially expressed circRNAs. The upper panel shows the expression density of the top 10 upregulated and downregulated circRNAs in the samples, with five quantiles and the mean line displayed. The lower panel shows the expression heatmap of the top 10 upregulated and downregulated circRNAs in the samples. E shows the predicted binding sites of CST7 and hsa-miR-3130-5p. F shows the predicted binding sites of CST7 and hsa-miR-18a-3p. The E / F predictions are based on Targetscan analysis (https: / / www.targetscan.org / vert_80 / ), which displays the binding regions and binding strengths of miRNAs to mRNAs. Data are presented as mean ± SD for the HCM group (n = 8) and the control group (n = 8). Data are presented as mean ± SD for the n = 6 group. * indicates P < 0.05, * indicates P < 0.01, and * indicates P < 0.001.

[0057] Figure 8 This is a dual-fluorescence staining experiment of hsa-miR-18a-3p targeting CST7 in rat cardiomyocytes H9C2, n=10.

[0058] Figure 9 This is a dual-fluorescence staining experiment of hsa-miR-3130-5p targeting CST7 in rat cardiomyocytes H9C2, n=10. DETAILED DESCRIPTION

[0059] In order to help those skilled in the art better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below in conjunction with the drawings in the embodiments of this specification. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this specification, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of this application.

[0060] Figure 1 The present invention provides a schematic flow chart of a computer-assisted drug screening method based on CST7 and miRNA, which specifically includes:

[0061] S101: Acquire CST7 gene data and miRNA gene data.

[0062] In some embodiments of the present invention, the miRNA gene data is hsa-miR-18a-3p and / or hsa-miR-3130-5p gene data.

[0063] In some embodiments of the present invention, the gene data includes molecular sequence information data, molecular structure data, and binding site data.

[0064] In some embodiments of the present invention, the CST7 gene encodes a protein called Cystatin F, which is a cysteine ​​protease inhibitor. CST7 plays a role in a variety of physiological and pathological processes, including immune responses, inflammation, and healthy and diseased states of the nervous system. CST7 is expressed in natural cytotoxic (NK) cells and CD8+ T cells. In the context of Alzheimer's disease (AD), CST7 expression in microglia is significantly upregulated. CST7 is also associated with the differentiation of Th2 cells in chronic hepatitis B virus (HBV) infection, which may promote the development of chronic HBV infection.

[0065] In some embodiments of the present invention, hsa-miR-18a-3p is a specific microRNA (miRNA) that plays a role in various biological processes. hsa-miR-18a-3p is downregulated in patients with anterior cruciate ligament osteoarthritis (OA), improving cartilage matrix remodeling and suppressing inflammatory responses, thereby playing a role in OA. miR-18a-5p (closely related to hsa-miR-18a-3p) acts as an oncogene in non-small cell lung cancer (NSCLC).

[0066] In some embodiments of the present invention, hsa-miR-3130-5p is a microRNA (miRNA) that plays a role in cell differentiation, proliferation, apoptosis, and metabolism, especially in cancer, cardiovascular disease, and neurodegenerative diseases.

[0067] In some embodiments of the present invention, to explore differentially expressed genes in HCM, we performed transcriptome analysis on our own sequencing data and the public dataset GSE141910 to identify differentially expressed genes between HCM patients and normal controls, and performed intersection analysis with macrophage-related genes. In the self-sequencing data, 866 differentially expressed genes were screened, of which 309 genes were upregulated and 557 genes were downregulated ( Figure 4 A), red dots represent up-regulated genes, blue dots represent down-regulated genes, and gray dots represent genes with no significant differences. The heat map shows the 10 most significantly up- and down-regulated genes, with CST7 specifically marked.

[0068] In some embodiments of the present invention, in the GSE141910 dataset, 3426 differentially expressed genes were obtained by using the limma package analysis, of which 2144 genes were upregulated and 1282 genes were downregulated ( Figure 4 B) Yellow dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes with no significant difference in expression. The heat map shows the 10 most significantly upregulated and downregulated genes, with CST7 specifically annotated.

[0069] In some embodiments of the present invention, 53 differentially expressed genes with consistent expression trends were screened by taking the intersection of up-regulated genes and down-regulated genes in the two datasets ( Figure 4 C), blue represents up-regulated genes in our center's own sequencing dataset, orange represents down-regulated genes in the GSE141910 dataset, light pink represents up-regulated genes in the GSE141910 dataset, and dark pink represents down-regulated genes in our center's own sequencing dataset.

[0070] In some embodiments of the present invention, to further screen key HCM genes related to macrophages, we performed intersection analysis on these 53 differentially expressed genes and 240 known macrophage-related genes, and identified three potential candidate genes: CX3CR1, IL31RA, and CST7 ( Figure 4 D), pink represents 53 differentially expressed genes with consistent expression trends, blue represents MRGs, and the intersection genes of the three are marked with different colors.

[0071] In some embodiments of the present invention, CST7 showed significant high expression in the HCM group in GSE141910 and our center's own sequencing data set ( Figure 4 E), the horizontal axis is the gene name, the vertical axis is the expression level, the red column represents the HCM group, and the blue column represents the control group.

[0072] In some embodiments of the present invention, in order to explore the causal relationship between prognostic candidate genes and HCM, we used the Mendelian randomization (MR) method to screen out genes significantly associated with HCM. First, we used the inverse variance weighted (IVW) method to perform MR analysis on the three genes Cx3cr1, Il31ra, and Cst7 screened in previous studies. The results showed that CST7, as a protective factor (OR < 1, P < 0.05), had a significant causal association with HCM; and CX3CR1, as a risk factor (OR > 1, P < 0.05), was also significantly associated with HCM. Given that the P value of CST7 was more significant, we focused on CST7 in subsequent studies ( Figure 5 A).

[0073] In some embodiments of the present invention, a scatter plot is drawn to show the effects of CST7-related single nucleotide polymorphisms (SNPs) on exposure (CST7 expression) and outcome (HCM). The IVW fitting results show that the slope of CST7 is negative, further confirming that it is a protective factor ( Figure 5 B) If there is an intercept in the graph, it suggests that there may be the influence of confounding factors.

[0074] In some embodiments of the present invention, the forest plot shows that the effects of each SNP are mainly concentrated on the left side, supporting the protective effect of CST7 ( Figure 5 C) Each horizontal solid line reflects the result estimated by the Wald ratio method for a single SNP. The solid line is completely to the left of 0, indicating that the result estimated by this SNP is that an increase in the exposure factor can reduce the risk of the outcome variable; the solid line is completely to the right of 0, indicating that the result estimated by this SNP is that an increase in the exposure factor can increase the risk of the outcome variable; and those crossing 0 indicate that the results are not significant, with the SNP point on the left indicating a decrease (safety factor) and the SNP point on the right indicating an increase (risk factor); the bottom blue line represents the overall effect of Inverse variance weighted, with CST7 being the safety factor for the outcome (SNP point on the left).

[0075] In some embodiments of the present invention, the funnel plot is bilaterally symmetrical, which is consistent with Mendel's second law, indicating that the exposure factor is less affected by the confounding factor ( Figure 5 D). The horizontal axis represents the beta value of the instrumental variable, indicating the effect of the SNP on CST7 gene expression; the vertical axis represents the inverse of the standard error of the instrumental variable, reflecting the precision of the estimate. The symmetric distribution of points in the figure indicates the robustness of the results; deviations from symmetry may indicate bias.

[0076] In some embodiments of the present invention, the results of the sensitivity analysis showed that the Q_pval of the heterogeneity test was greater than 0.05, indicating that there was no significant heterogeneity between samples; the P val of the horizontal pleiotropy test was also greater than 0.05, indicating that there were no significant pleiotropy or confounding factors in the study ( Figure 5 E), Q_pval is the result of heterogeneity test, and Pval is the result of horizontal pleiotropy test.

[0077] In some embodiments of the present invention, in order to evaluate the therapeutic effect of AAV-mediated macrophage-specific CST7 gene overexpression in the HCM mouse model, we constructed an AAV6 vector driven by the macrophage-specific promoter F4 / 80, carried the CST7 gene for overexpression, and delivered it to the HCM mouse model by multi-point in situ injection into the myocardium.

[0078] In some embodiments of the present invention, RT-qPCR results showed that the expression of CST7 in the heart of HCM mice was significantly increased, and the CST7 mRNA level in the CST7 gene therapy group was also significantly increased compared with the NC group ( Figure 6 A).

[0079] In some embodiments of the present invention, HE staining was used to observe changes in the cardiac tissue structure of mice. In the normal group, the endocardium and epicardium were intact, and the myocardial cells were tightly arranged. In the HCM group, the myocardial cell gaps were widened (black arrows), fibroblasts proliferated (red arrows), vacuoles appeared in the myocardial cell cytoplasm (blue arrows), a small amount of congestion in the blood vessels (yellow arrows), and insoluble fibrin, red blood cells and flavonoids were deposited in the cardiac cavity (orange arrows). The structure of the CST7 treatment group was significantly restored, and the pathological manifestations were significantly alleviated. Therefore, HE staining observations showed that the HCM group had obvious lesions in the cardiac structure, and CST7 gene therapy significantly alleviated the abnormal HE staining of the myocardium ( Figure 6 B).

[0080] In some embodiments of the present invention, WGA (green) staining was used to assess cardiomyocyte size, and DAPI (blue) staining was used to visualize cell nuclei. Cardiomyocytes in the HCM group were significantly hypertrophic, as evidenced by thickened fibers and increased size. Cardiomyocyte hypertrophy was significantly reduced in the CST7-treated group, with myocardial fiber size approaching normal. Therefore, WGA staining results showed that cardiomyocytes in the HCM group were significantly hypertrophic, and the CST7-treated group significantly reduced myocardial hypertrophy compared to the control group ( Figure 6 C).

[0081] In some embodiments of the present invention, Masson staining is used to detect myocardial fibrosis, with fibrotic areas marked in blue. Myocardial fibrosis was evident in the HCM group, as evidenced by the deposition of a large amount of collagen fibers. Red or purple areas were visible in the fibrotic tissue, representing the proliferation and deposition of collagen fibers. Collagen fiber deposition was significantly reduced in the CST7-treated group, and the degree of fibrosis was reduced. Therefore, further Masson staining showed that the HCM group had significant collagen fiber deposition, and CST7 treatment significantly reduced the degree of myocardial fibrosis ( Figure 6 D).

[0082] In some embodiments of the present invention, the heart size and morphology of the normal group mice were normal. The heart of the HCM group was significantly enlarged, showing typical features of hypertrophic cardiomyopathy. The heart size of the CST7-treated group was close to normal, and the cardiac hypertrophy was significantly alleviated. Therefore, macroscopic observation of heart size revealed that the heart of the HCM group was significantly enlarged, while the CST7-treated group effectively suppressed myocardial enlargement ( Figure 6 E).

[0083] In some embodiments of the present invention, the electrocardiogram of the HCM group showed a significant increase in R wave voltage, which was manifested as a sharp peak in the R wave. The R wave voltage of the CST7-treated group was significantly reduced, and the R wave peak phenomenon was significantly alleviated. Therefore, the electrocardiogram results showed that the R wave voltage of the HCM group was significantly increased, and Cst7 treatment significantly alleviated the R wave peak phenomenon ( Figure 6 F).

[0084] In some embodiments of the present invention, ultrasound examination results showed that the ventricular septum thickness in the HCM group was significantly increased, consistent with the characteristics of hypertrophic cardiomyopathy. The ventricular septum thickness in the CST7 treatment group was significantly reduced compared with the HCM group, approaching normal levels. Therefore, cardiac ultrasound examination showed that the ventricular septum thickness in the HCM group was significantly thickened, while the ventricular septum thickness in the CST7 treatment group was significantly reduced ( Figure 6 G).

[0085] In some embodiments of the present invention, F4 / 80 promoter-mediated AAV6 delivery of CST7 showed significant therapeutic effects in reducing myocardial hypertrophy and fibrosis and improving cardiac dysfunction in mice with hypertrophic cardiomyopathy.

[0086] S102: Select the sequence information and spatial structure of the CST7 gene and the sequence information and spatial structure of the miRNA gene, and determine the binding sites of the two as the binding sites of the targeted drug.

[0087] In some embodiments of the present invention, to perform differential expression analysis, the applicant collected myocardial tissue samples from patients with myocardial hypertrophy (HCM) (n=12) and a healthy control group (n=12). After extracting total RNA, miRNA and ccRNA were sequenced using high-throughput sequencing technologies (such as the Illumina platform). The resulting raw data first underwent quality control to remove low-quality reads and adapter sequences. The clean data were then aligned to a reference genome using a standardized bioinformatics analysis pipeline. The DESeq2 software package was used to perform differential expression analysis on the miRNA and ccRNA expression levels between the two groups of samples. A threshold of |log2 (fold change)|>1 and a p-value <0.05 was set to screen for significantly differentially expressed miRNAs and ccRNAs.

[0088] In some embodiments of the present invention, to investigate the differential expression of miRNAs and circRNAs between HCM and control groups, we performed differential expression analysis on self-sequencing data generated by our center, comparing samples from the disease group and the control group. The results showed that a total of 71 differentially expressed miRNAs were identified, of which 34 were upregulated in the HCM group and 37 were downregulated (see volcano plot, Figure 7 A). Through heat map ( Figure 7 B) shows the top 10 miRNAs with the highest up- and down-regulated folds. In terms of circRNAs, a total of 1542 differentially expressed circRNAs were found, of which 597 were up-regulated and 945 were down-regulated in the HCM group (see volcano plot, Figure 7 C). Similarly, the heat map ( Figure 7 D) Shows the top 10 circRNAs with the highest up- and down-regulated folds.

[0089] In some embodiments of the present invention, to explore the potential regulatory relationship between differentially expressed miRNAs and the target gene CST7, we used the TargetScan (Version 7.2) database to predict miRNA-mRNA binding sites. The identified differentially expressed miRNA sequences were input into TargetScan and analyzed for potential binding sites within the CST7 3'UTR region. The results indicated that the upregulation of CST7 expression may be regulated by two miRNAs: hsa-miR-18a-3p and hsa-miR-3130-5p. Figure 7 E is the predicted binding site of CST7 and hsa-miR-3130-5p, Figure 7 F is the predicted binding site of CST7 and hsa-miR-18a-3p. These results reveal the potential regulatory mechanism of CST7 in HCM and provide a basis for further study of its function.

[0090] In some embodiments of the present invention, to validate the direct regulatory effects of hsa-miR-3130-5p and hsa-miR-18a-3p on CST7, we conducted a dual-luciferase reporter gene assay. First, a fragment encompassing the 3'UTR region of the CST7 gene (containing a predicted miRNA binding site) was cloned into the pGL3-promoter vector to construct a wild-type (WT) reporter gene vector. Key bases in the miRNA binding site were altered through site-directed mutagenesis to construct a mutant (MUT) reporter gene vector. Next, rat cardiomyocytes (H9C2) were cultured in 96-well plates. When the cell density reached 70%-80%, the WT or MUT vectors were co-transfected with a miRNA mimic or negative control (NC) using Lipofectamine 3000 reagent. Forty-eight hours later, the activities of firefly and Renilla luciferase were measured using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions. Firefly luciferase activity was used to reflect the expression level of the reporter gene, and Renilla luciferase activity was used as an internal control for normalization. Three replicates were set up for each group of experiments. The experimental results are expressed as mean ± standard deviation, and statistical analysis was performed using t-test. The results showed that hsa-miR-18a-3p and hsa-miR-3130-5p could significantly reduce the luciferase activity of the WT vector, but had no significant effect on the MUT vector ( Figure 8 、 Figure 9 ).

[0091] S103: Using a computer-assisted drug screening method to obtain candidate drugs targeting the binding site.

[0092] In some specific embodiments of the present invention, computer-assisted drug screening is a technology that utilizes computer-assisted drug design methods to screen drugs. It can help researchers quickly screen a large number of small molecule compounds for candidate drugs that have strong binding ability to target proteins and have potential efficacy.

[0093] In some specific embodiments of the present invention, the method process of computer-assisted drug screening is: obtaining the binding site of the CST7 gene and the miRNA gene; screening compounds with similar sequences and structures in the molecular database based on the sequence information and spatial structure of the binding site of the CST7 gene and the miRNA gene; performing molecular docking between the screened compounds and the CST7 gene to calculate the binding energy score of the target compound, and sorting according to the score to obtain candidate drugs.

[0094] In some specific embodiments of the present invention, the method process of computer-assisted drug screening is: obtaining the binding site of the CST7 gene and the miRNA gene and the spatial structure of the complex after binding; screening structurally complementary compounds in the molecular database based on the binding site of the CST7 gene and the miRNA gene and the spatial structure of the complex after binding; performing molecular docking on the screened compounds and the complex after binding of the CST7 gene and the miRNA gene, calculating the binding energy score of the target compound, and sorting according to the score to obtain candidate drugs.

[0095] In some specific embodiments, in the molecular docking, affinity refers to the tightness of the binding between the molecule and the receptor. High affinity means that the binding is more stable, while low affinity means that the binding is less stable. Affinity is usually obtained by calculation, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd). In order to evaluate affinity, some scoring systems or scoring methods are usually used to quantify the interaction between the molecule and the receptor. These scoring methods are based on different algorithms and physical models and can reflect the binding energy, interaction type and affinity between the molecule and the receptor.

[0096] In one embodiment of the present application, the training set data is input into TargetScan, a widely used bioinformatics database, which helps researchers understand the role of microRNAs (miRNAs) in gene expression regulation by predicting their target genes. The database predicts potential target genes of miRNAs by using the complementary pairing of the conserved seed region (usually the 2nd to 8th nucleotides of the miRNA) and the 3' untranslated region (3'UTR) of the messenger RNA (mRNA).

[0097] Based on the same idea, the embodiment of the present invention also provides Figure 2 , a schematic diagram of a computer-assisted drug screening system based on CST7 and miRNA, specifically including.

[0098] Data acquisition unit: acquires CST7 gene data and miRNA gene data.

[0099] Data processing unit: select the sequence information and spatial structure of the CST7 gene and the sequence information and spatial structure of the miRNA gene, and determine the binding sites of the two as the binding sites of the targeted drug.

[0100] Drug screening unit: A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site. The computer-assisted drug screening method is the computer-assisted drug screening method among the computer-assisted drug screening methods described above.

[0101] The foregoing description of specific embodiments of this specification is intended to be a description of other embodiments. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps recited in the claims may be performed in a different order than that described in the embodiments and still achieve the desired results. Furthermore, the processes depicted in the accompanying drawings do not necessarily require the specific order shown or the sequential order to achieve the desired results. In certain embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0102] Figure 3 A schematic diagram of a computer-assisted drug screening device based on CST7 and miRNA provided in an embodiment of the present invention specifically includes: a memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the computer-assisted drug screening method described above is implemented.

[0103] The various embodiments in this specification are described in a progressive manner. Similar portions between the various embodiments can be referenced to each other, and each embodiment focuses on the differences between the other embodiments. In particular, the device, electronic device, and non-volatile computer storage medium embodiments are generally similar to the method embodiments, so their descriptions are relatively simplified. For relevant details, refer to the descriptions of the method embodiments.

[0104] The apparatus, electronic device, and non-volatile computer storage medium provided in the embodiments of this specification correspond to the method. Therefore, the apparatus, electronic device, and non-volatile computer storage medium also have similar beneficial technical effects as the corresponding method. Since the beneficial technical effects of the method have been described in detail above, the beneficial technical effects of the corresponding apparatus, electronic device, and non-volatile computer storage medium will not be repeated here.

[0105] The various embodiments in this specification are described in a progressive manner. Similar parts between the various embodiments can be referred to in conjunction with each other. Each embodiment focuses on the differences between the other embodiments. In particular, the system embodiments are generally similar to the method embodiments, so the description is relatively simple. For relevant parts, refer to the description of the method embodiments.

[0106] The foregoing is merely an embodiment of the present invention and is not intended to limit the present application. For those skilled in the art, various modifications and variations may be made to the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included within the scope of the claims of the present application.

Claims

1. A computer-assisted drug screening method based on CST7 and miRNA, characterized in that: The drug is used to treat hypertrophic cardiomyopathy, and the method specifically comprises: Obtain CST7 gene data and miRNA gene data; The sequence information and spatial structure of the CST7 gene and the sequence information and spatial structure of the miRNA gene are selected to determine the binding sites of the two as the binding sites of the targeted drug; A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site; The miRNA gene is hsa-miR-18a-3p and / or hsa-miR-3130-5p.

2. The computer-assisted drug screening method according to claim 1, wherein the computer-assisted drug screening method comprises: Obtain the binding sites of CST7 gene and miRNA gene; Based on the sequence information and spatial structure of the binding sites between the CST7 gene and the miRNA gene, compounds with similar sequences and structures were screened in the molecular database; Molecular docking is performed between the screened compounds and the CST7 gene to calculate the binding energy scores of the targeted compounds, and candidate drugs are obtained by ranking according to the scores.

3. The computer-assisted drug screening method according to claim 1, wherein the computer-assisted drug screening method comprises: Obtain the binding sites of CST7 gene and miRNA gene and the spatial structure of the complex after binding; Based on the binding sites of CST7 gene and miRNA gene and the spatial structure of the complex after binding, compounds with complementary structures were screened in the molecular database; Molecular docking is performed on the screened compounds and the complex formed by binding of the CST7 gene and the miRNA gene, the binding energy scores of the targeted compounds are calculated, and candidate drugs are obtained by ranking according to the scores.

4. The computer-aided drug screening method according to claim 2, wherein the compound comprises a protein analog, an antibody, DNA, or RNA.

5. A computer-assisted drug screening system based on CST7 and miRNA, comprising: Data acquisition unit: acquiring CST7 gene data and miRNA gene data, wherein the miRNA gene is hsa-miR-18a-3p and / or hsa-miR-3130-5p; Data processing unit: selects the sequence information and spatial structure of the CST7 gene and the sequence information and spatial structure of the miRNA gene, and determines the binding sites of the two as the binding sites of the targeted drug; Drug screening unit: A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site. The computer-assisted drug screening method is the computer-assisted drug screening method according to any one of claims 1 to 4.

6. A computer-assisted drug screening device based on CST7 and miRNA, comprising: a memory and a processor, wherein the memory is used to store program instructions; The processor is used to call program instructions, and when the program instructions are executed, the computer-assisted drug screening method according to any one of claims 1 to 4 is implemented.

7. A computer-readable storage medium having a computer program thereon, comprising: When the computer program is executed by a processor, the computer-assisted drug screening method according to any one of claims 1 to 4 is implemented.

8. A drug obtained by screening using the computer-assisted drug screening method according to any one of claims 1 to 4.

Images