Screening method and application of microRNA combination in atherosclerosis medicine

Abstract

The invention belongs to the field of cardiovascular atherosclerosis, and particularly relates to a screening method and application of a microRNA combination in atherosclerosis drugs, and the screening method comprises the following steps: S1, setting a cell experiment control group; s2, constructing an endothelial cell senescence model; s3, detecting the expression quantity of the P53 protein; s4, constructing an endothelial cell injury model for overexpressing the microRNA; and S5, evaluating the action effect of the microRNA combination. According to the invention, the microRNA overexpression endothelial cell injury model is constructed to simulate prevention or treatment of atherosclerosis, so that effective screening of the microRNA combination for preventing or treating atherosclerosis is realized, a theoretical basis is provided for developing a new treatment strategy and medicine, and a new thought and method are provided for early treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a screening method and application of a microRNA combination used in atherosclerosis drugs. Background Art

[0002] Atherosclerosis is a chronic inflammatory disease that mainly affects arterial blood vessels. It is caused by the deposition of cholesterol and other lipid substances on the inner wall of the artery to form plaques, which causes the blood vessel wall to become hardened and narrow. Atherosclerosis is the main cause of myocardial infarction and stroke. Current research indicates that aging of vascular endothelial cells is the key to the onset of cardiovascular disease. Endothelial cell aging is closely related to VEC dysfunction and atherosclerotic plaque formation. When plaques rupture or ulcerate, they can trigger platelet aggregation and thrombosis. If this happens in the coronary arteries, it can lead to myocardial infarction. Similarly, when plaques rupture or thrombosis forms in the cerebral arteries, it can lead to stroke.

[0003] At present, although researchers have gradually deepened their understanding of endothelial cell aging, the drugs and targets that can be used to prevent and treat endothelial cell aging are still relatively limited. Endothelial cell aging is an important factor in cardiovascular diseases such as atherosclerosis. Therefore, it is of great significance to find new drugs and targets to delay the aging of endothelial cells. By discovering more drugs and targets, the process of endothelial cell aging can be effectively delayed, thereby providing more options and opportunities for the prevention and treatment of cardiovascular diseases. Therefore, we proposed a screening method for screening microRNA combinations in the preparation of drugs for the prevention or treatment of atherosclerosis. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to provide a method for screening microRNA combinations for atherosclerosis drugs. By constructing an endothelial cell injury model that overexpresses microRNA to simulate the prevention or treatment of atherosclerosis, effective screening of microRNA combinations for the prevention or treatment of atherosclerosis is achieved, providing new ideas and methods for the early treatment of atherosclerosis.

[0005] A second object of the present invention is to provide a method for screening a microRNA combination in an atherosclerosis drug and its application in preparing a drug for preventing or treating atherosclerosis.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention discloses a method for screening a microRNA combination in an atherosclerosis drug, comprising the following steps:

[0008] S1: Setting a cell experiment control group: the cell experiment control group is a microRNA combination, and each microRNA combination consists of at least two microRNAs;

[0009] S2: Construction of endothelial cell aging model: Human umbilical vein endothelial cells were prepared into a cell suspension and inoculated into a 96-well plate for culture. After adding hydrogen peroxide, the cell survival rate was calculated to establish the optimal injury model conditions;

[0010] S3: Detecting the expression of P53 protein: Perform WB detection on the cell experimental control group in step S1 to determine the effect of the microRNA combination on the expression of P53 protein, and select the microRNA combination with the greatest difference as the preferred experimental control group;

[0011] S4: constructing an endothelial cell injury model overexpressing microRNA: constructing an endothelial cell injury model overexpressing microRNA based on the preferred experimental control group selected in step S3 and the optimal injury model conditions established in step S2;

[0012] S5: Evaluation of the effect of the microRNA combination: The normal endothelial cell group, the endothelial cell aging model group, the normal endothelial cell group overexpressing microRNA, and the endothelial cell aging model group overexpressing microRNA were tested to evaluate the effect of the microRNA combination.

[0013] Preferably, in step S1, the microRNA includes miR-34a, miR-449a and miR-16.

[0014] Preferably, in step S2, 2-8×10 human umbilical vein endothelial cells in logarithmic growth phase are prepared 4 100uL of the cell suspension of cells / mL was inoculated into a 96-well plate. After culturing for 24 hours, the medium was replaced with serum-free culture medium. Hydrogen peroxide was added to stimulate the cells, and the cell survival rate was calculated to establish the optimal injury model conditions.

[0015] Through the above technical scheme, a cell model with aging characteristics (endothelial cell aging model) can be prepared, and subsequent experiments can be carried out in serum-free culture medium, so as to evaluate the effects of the microRNA combination on aging cells, including changes in indicators such as cell viability, aging-related enzyme SA-β-Gal positivity rate, cell structure, and aging protein expression, thereby revealing the regulatory effect of the microRNA combination on vascular endothelial cell aging, and providing an experimental basis for studying the pathogenesis and treatment of atherosclerosis.

[0016] Preferably, the concentration of hydrogen peroxide is 50-400 umol / L, and the stimulation time is 2-8 hours.

[0017] More preferably, the concentration of hydrogen peroxide is 50umol / L, 100umol / L, 150umol / L, 200umol / L and 400umol / L, and the stimulation time is 2 hours, 4 hours, 6 hours and 8 hours.

[0018] Further preferably, adding hydrogen peroxide to stimulate the cells is as follows: stimulating the cells with 50umol / L, 100umol / L, 150umol / L, 200umol / L and 400umol / L hydrogen peroxide in serum-free culture medium for 4 hours, and stimulating the cells with 100umol / L hydrogen peroxide for 2 hours, 4 hours, 6 hours and 8 hours, respectively, to obtain the optimal injury model conditions (optimal hydrogen peroxide concentration and stimulation time).

[0019] Through the above technical scheme, we can study the degree and changes of cell aging under different stimulation times, evaluate the degree and changes of cell aging under different concentrations and stimulation times, and the regulatory effect of microRNA combination on vascular endothelial cell aging at different aging degrees and times. This can provide a deep understanding of the mechanism of action of microRNA combination in the aging process and provide experimental basis for finding new strategies for preventing and treating atherosclerosis.

[0020] Preferably, before calculating the cell viability, 20uL of cell viability assay reagent is added to each well of a 96-well plate and incubated at 37°C for 2 hours; the cell viability is calculated using an automatic microplate reader, which measures the absorbance of each replicate well in each experimental group at a wavelength of 490nm.

[0021] Through the above technical scheme, the survival rate data of cells at different concentrations and stimulation times can be obtained, so as to evaluate the toxicity of hydrogen peroxide to cells and the response of cells to its stimulation. This can further understand the degree of damage to cells caused by hydrogen peroxide and the adaptability of cells, and provide experimental basis for further studying the mechanism of cell aging and finding strategies to prevent and treat aging-related diseases.

[0022] Preferably, in step S3, the step of determining the effect of the microRNA combination on the expression level of P53 protein is:

[0023] (1) Lysing the cells of the experimental control group using a protein extraction solution and extracting proteins;

[0024] (2) determining the protein concentration in (1) using a protein determination kit;

[0025] (3) adding the protein from (1) to SDS-PAGE gel for protein separation;

[0026] (4) transferring the protein separated in (3) to a PVDF membrane, and placing the PVDF membrane in a blocking buffer;

[0027] (5) incubating the PVDF membrane treated in (4) with P53 antibody, and then washing with washing buffer;

[0028] (6) incubating the PVDF membrane treated in (5) with an HRP-labeled secondary antibody, and then washing with a washing buffer;

[0029] (7) adding ECL reagent to the PVDF membrane treated in (6) to make the P53 protein emit light;

[0030] (8) Taking a luminescent image of the PVDF membrane treated in (7) and determining the expression level of P53 protein by image analysis.

[0031] Preferably, in step S5, the normal endothelial cell group is untreated normal endothelial cells; the endothelial cell aging model group is endothelial cells induced to age by hydrogen peroxide; the normal endothelial cell group overexpressing microRNA is endothelial cells overexpressing microRNA in normal endothelial cells, and the endothelial cell aging model group overexpressing microRNA is endothelial cells in which endothelial cells overexpressing microRNA are exposed to hydrogen peroxide.

[0032] Preferably, in step S5, the detection includes cell aging detection, cell viability detection, cell cycle detection and protein expression detection.

[0033] Preferably, the cell aging detection adopts β-galactosidase staining method, the cell viability detection adopts CCK-8 method, the cell cycle detection adopts flow cytometer analysis, and the protein expression detection adopts WB detection.

[0034] Preferably, the target proteins for protein expression detection include P16, P21 and phosphorylated histone H2A.X.

[0035] By testing cells in different groups, we can fully understand the aging degree, vitality state, cell cycle and expression of specific proteins of the grouped cells, which will help reveal the mechanism of microRNA overexpression on endothelial cell aging, deeply understand the regulatory process of cell aging, and provide a theoretical basis for the treatment and intervention of related diseases. In-depth research on the mechanism of action of microRNA combinations in atherosclerosis will provide a theoretical basis for the development of new treatment strategies and drugs, and provide new ideas and methods for the early prevention and treatment of atherosclerosis.

[0036] The second aspect of the present invention discloses the use of any of the above-described methods for screening microRNA combinations in atherosclerosis drugs in the preparation of drugs for preventing or treating atherosclerosis.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention stimulates cells by hydrogen peroxide to simulate endothelial cell damage in atherosclerosis and establishes optimal damage model conditions; by detecting the expression amount of P53 protein, the regulatory effect of microRNA combination on P53 protein expression can be evaluated, and a suitable microRNA combination can be selected as a research object; by constructing an endothelial cell damage model overexpressing microRNA to simulate the effect of preventing or treating atherosclerosis, by performing CCK-8 method detection on cell viability of cells in different groups, SA-β-Gal staining to identify cell senescence, flow cytometer analysis of cell cycle and protein expression detection, the preventive or therapeutic effect of microRNA combination on atherosclerosis is evaluated, a theoretical basis is provided for the development of new treatment strategies and drugs, and new ideas and methods are provided for the early prevention and treatment of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the process of screening the microRNA combination of the present invention;

[0040] Figure 2 It is a schematic diagram of the process of determining the expression amount of P53 protein by the microRNA combination in the screening method of the microRNA combination of the present invention;

[0041] Figure 3 This is a schematic diagram of cell viability of four groups of cell experimental control groups in Example 2 of the present invention;

[0042] Figure 4 Schematic diagram of the cell ultrastructure of the four groups of cell experimental control groups in Example 2 of the present invention under a transmission electron microscope (1 μm);

[0043] Figure 5 It is a schematic diagram of the detection method for evaluating the effect of the microRNA combination in the screening method of the present invention. DETAILED DESCRIPTION

[0044] Below, in conjunction with the accompanying drawings and specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0045] Example 1

[0046] A method for screening microRNA combinations in atherosclerosis drugs, such as Figure 1 As shown, the following steps are included:

[0047] S1: Setting a cell experiment control group: the cell experiment control group is a microRNA combination, and each microRNA combination consists of at least two microRNAs;

[0048] S2: Construction of endothelial cell aging model: Human umbilical vein endothelial cells were prepared into a cell suspension and inoculated into a 96-well plate for culture. After adding hydrogen peroxide, the cell survival rate was calculated to establish the optimal injury model conditions;

[0049] S3: Detecting the expression of P53 protein: Perform WB detection on the cell experimental control group in step S1 to determine the effect of the microRNA combination on the expression of P53 protein, and select the microRNA combination with the greatest difference as the preferred experimental control group;

[0050] S4: constructing an endothelial cell injury model overexpressing microRNA: constructing an endothelial cell injury model overexpressing microRNA based on the preferred experimental control group selected in step S3 and the optimal injury model conditions established in step S2;

[0051] S5: Evaluation of the effect of the microRNA combination: The normal endothelial cell group, the endothelial cell aging model group, the normal endothelial cell group overexpressing microRNA, and the endothelial cell aging model group overexpressing microRNA were tested to evaluate the effect of the microRNA combination.

[0052] More specifically, the screening method in this embodiment comprises the following specific steps:

[0053] S1: Setting up a cell experiment control group: The cell experiment control group is a microRNA combination, each microRNA combination is composed of at least two microRNAs, and the endothelial cells are cultured to 80% confluence and then transfected using a kit;

[0054] S2: Construction of endothelial cell aging model: Human umbilical vein endothelial cells were prepared into 2-8×10 cells using the basic culture medium DMEM / F12 at a culture temperature of 37°C, a CO2 concentration of 5%, and a humidity of 95%. 4 The cells / mL cell suspension was inoculated in a 96-well plate for culture. After adding hydrogen peroxide, the cell survival rate was calculated to establish the optimal injury model conditions.

[0055] S3: Detecting the expression of P53 protein: Perform WB detection on the cell experimental control group in step S1 to determine the effect of the microRNA combination on the expression of P53 protein, and select the microRNA combination with the greatest difference as the preferred experimental control group;

[0056] S4: constructing an endothelial cell injury model overexpressing microRNA: constructing an endothelial cell injury model overexpressing microRNA based on the preferred experimental control group selected in step S3 and the optimal injury model conditions established in step S2;

[0057] S5: Evaluation of the effect of the microRNA combination: The normal endothelial cell group, the endothelial cell aging model group, the normal endothelial cell group overexpressing microRNA, and the endothelial cell aging model group overexpressing microRNA were tested to evaluate the effect of the microRNA combination.

[0058] Example 2

[0059] A method for screening microRNA combinations in atherosclerosis drugs, comprising the following steps:

[0060] S1: Set up cell experimental control groups: 4 cell experimental control groups are microRNA combinations, each microRNA combination is composed of at least two microRNAs, and the 4 cell experimental control groups are "miR-34a, miR-449a", "miR-449a, miR-16", "miR-34a, miR-449a, miR-16" and "miR-34a, miR-16". Human umbilical vein endothelial cells are cultured to 80% confluence and then transfected using a kit.

[0061] S2: Construction of endothelial cell aging model: Human umbilical vein endothelial cells in the logarithmic growth phase were used to make 2-8×10 4 / mL cell suspension, 100uL of cell suspension was inoculated in a 96-well plate, 6 replicates were set for each group, and the serum-free culture medium was replaced after 24 hours of culture, and the cells were stimulated with 0, 50umol / L, 100umol / L, 150umol / L, 200umol / L and 400umol / L hydrogen peroxide for 2 hours, 4 hours, 6 hours and 8 hours respectively to obtain the best injury model conditions (optimal hydrogen peroxide concentration and stimulation time). After treatment, the old culture medium was discarded, and 20uL of cell viability assay reagent was added to each well of the 96-well plate, and incubated at 37°C for 2 hours. The absorbance value of each experimental group was measured at a wavelength of 490nm by an automatic microplate reader, and the cell survival rate was calculated to establish the best injury model conditions.

[0062] S3: Detection of the expression of P53 protein: The four cell experimental control groups "miR-34a, miR-449a", "miR-449a, miR-16", "miR-34a, miR-449a, miR-16" and "miR-34a, miR-16" obtained in step S1 were subjected to WB detection respectively. The flow chart of detecting the expression of P53 protein in the four cell experimental control groups is shown as follows Figure 2 As shown, the cell viability diagram of the four groups of cell experimental control groups is as follows Figure 3 The schematic diagram of the cell ultrastructure of the four groups of cell experimental control groups under transmission electron microscope is shown in Figure 4 As shown. By comparing the effects of the microRNA combinations of the four cell experimental control groups on the expression of P53 protein, the microRNA combination with the greatest difference was selected as the preferred experimental control group. The specific steps are as follows:

[0063] (1) preparing a protein extract, and according to the instructions for use of the protein extract, lysing and extracting proteins from four cell experimental control group samples of "miR-34a, miR-449a", "miR-449a, miR-16", "miR-34a, miR-449a, miR-16" and "miR-34a, miR-16";

[0064] (2) preparing a protein assay kit, and measuring the protein concentration in each cell experimental control group using the protein assay kit;

[0065] (3) adding the same amount of protein from each cell experimental control group to the wells of SDS-PAGE gel for protein separation;

[0066] (4) Transferring the separated proteins to a PVDF membrane and placing the PVDF membrane in a blocking buffer to block nonspecific binding sites;

[0067] (5) incubating the PVDF membrane with the P53 antibody to allow it to bind to the target P53 protein, and then washing the PVDF membrane with a washing buffer to remove unbound antibodies;

[0068] (6) incubating the PVDF membrane with HRP-labeled secondary antibody to allow it to bind to the primary antibody bound to the P53 antibody;

[0069] (7) Wash the PVDF membrane again with washing buffer to remove unbound secondary antibodies, and add ECL reagent to make the P53 protein on the membrane emit light;

[0070] (8) The luminescent images on the PVDF membrane were captured using a chemiluminescence imaging system, and the expression level of P53 protein was determined using image analysis software.

[0071] S4: Construction of an endothelial cell injury model overexpressing microRNA: Based on the preferred experimental control group selected in step S3 and the optimal injury model conditions established in step S2, an endothelial cell injury model overexpressing microRNA is constructed. The endothelial cell aging model group overexpressing microRNA is to expose the endothelial cells overexpressing microRNA to a solution containing hydrogen peroxide to simulate the simultaneous presence of microRNA overexpression and endothelial cell aging.

[0072] S5: Evaluate the effect of microRNA combination: test the grouped normal endothelial cell group, endothelial cell aging model group, normal endothelial cell group overexpressing microRNA, and endothelial cell aging model group overexpressing microRNA. The endothelial cell aging model group is the endothelial cells of the optimal damage model conditions established in step S2; the normal endothelial cell group overexpressing microRNA is the endothelial cells of the preferred experimental control group selected in step S3; the endothelial cell aging model group overexpressing microRNA is the endothelial cells of the preferred experimental control group selected in step S3 exposed to the optimal damage model conditions established in S2 to simulate the situation of simultaneous microRNA overexpression and endothelial cell aging. By testing cells in different groups, such as Figure 5 As shown, the specific detection contents include using β-galactosidase (SA-β-gal) staining to identify whether the cells are senescent, using CCK-8 method to detect the viability of the cells, using flow cytometry to analyze the cell cycle, and using Western blot technology to detect the expression of proteins such as P16, P21, and phosphorylated histone H2A.X, in order to evaluate the preventive or therapeutic effects of the microRNA combination on atherosclerosis.

[0073] In summary, the screening method of the microRNA combination in the preparation of drugs for preventing or treating atherosclerosis can more accurately evaluate the preventive or therapeutic effect of the microRNA combination on atherosclerosis by setting up 4 experimental groups: "miR-34a, miR-449a", "miR-449a and miR-16", "miR-34a, miR-449a and miR-16", and "miR-34a and miR-16". By stimulating cells with hydrogen peroxide, endothelial cell damage in atherosclerosis can be simulated to establish the optimal damage model conditions. By detecting the expression level of P53 protein, the regulatory effect of the microRNA combination on endothelial cell aging can be evaluated, and Select appropriate microRNA combinations as research objects; and construct an endothelial cell injury model that overexpresses microRNA and divides the cells into groups: by overexpressing microRNA to simulate the effect of preventing or treating atherosclerosis, and dividing the cells into groups, it is helpful to compare the differences between different groups; and by detecting cells in different groups, the preventive or therapeutic effect of the microRNA combination on atherosclerosis can be evaluated, providing data support for further research, so that the present invention has the advantages of accurately evaluating and simulating atherosclerotic damage, screening suitable microRNA combinations, simulating preventive or therapeutic effects, and providing data support, etc., which solves the problem that there are few drugs and targets that can prevent and treat vascular endothelial cell aging.

[0074] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for screening microRNA combinations in atherosclerosis drugs, characterized in that: The steps include: S1: Setting a cell experiment control group: the cell experiment control group is a microRNA combination, and each microRNA combination consists of at least two microRNAs; S2: Construction of endothelial cell aging model: Human umbilical vein endothelial cells were prepared into cell suspension and inoculated into 96-well plates for culture. After adding hydrogen peroxide, the cell survival rate was calculated to establish the optimal injury model conditions; S3: Detecting the expression of P53 protein: Perform WB detection on the cell experimental control group in step S1 to determine the effect of the microRNA combination on the expression of P53 protein, and select the microRNA combination with the greatest difference as the preferred experimental control group; S4: constructing an endothelial cell injury model overexpressing microRNA: constructing an endothelial cell injury model overexpressing microRNA based on the preferred experimental control group selected in step S3 and the optimal injury model conditions established in step S2; S5: Evaluation of the effect of the microRNA combination: The normal endothelial cell group, the endothelial cell aging model group, the normal endothelial cell group overexpressing microRNA, and the endothelial cell aging model group overexpressing microRNA were tested to evaluate the effect of the microRNA combination.

2. The method for screening a microRNA combination in an atherosclerosis drug according to claim 1, characterized in that: In step S1, the microRNA includes miR-34a, miR-449a and miR-16.

3. The method for screening a microRNA combination in an atherosclerosis drug according to claim 1, characterized in that: In step S2, 2-8×10 human umbilical vein endothelial cells in logarithmic growth phase were prepared. 4 100uL of the cell suspension of cells / mL was inoculated into a 96-well plate. After culturing for 24 hours, the medium was replaced with serum-free culture medium. Hydrogen peroxide was added to stimulate the cells, and the cell survival rate was calculated to establish the optimal injury model conditions.

4. A method for screening a microRNA combination in an atherosclerosis drug according to claim 1 or 3, characterized in that: The concentration of the hydrogen peroxide is 50-400umol / L, and the stimulation time is 2-8 hours.

5. The method for screening a microRNA combination in an atherosclerosis drug according to claim 1, characterized in that: In step S3, the step of determining the effect of the microRNA combination on the expression level of P53 protein is as follows: (1) Lysing the cells of the experimental control group using a protein extraction solution and extracting proteins; (2) determining the protein concentration in (1) using a protein determination kit; (3) adding the protein from (1) to SDS-PAGE gel for protein separation; (4) transferring the protein separated in (3) to a PVDF membrane, and placing the PVDF membrane in a blocking buffer; (5) incubating the PVDF membrane treated in (4) with P53 antibody, and then washing with washing buffer; (6) incubating the PVDF membrane treated in (5) with an HRP-labeled secondary antibody, and then washing with a washing buffer; (7) adding ECL reagent to the PVDF membrane treated in (6) to make the P53 protein emit light; (8) Taking a luminescent image of the PVDF membrane treated in (7) and determining the expression level of P53 protein by image analysis.

6. The method for screening a microRNA combination in an atherosclerosis drug according to claim 1, characterized in that: In step S5, the normal endothelial cell group is untreated normal endothelial cells; the endothelial cell aging model group is endothelial cells induced to age by hydrogen peroxide; the normal endothelial cell group overexpressing microRNA is endothelial cells overexpressing microRNA in normal endothelial cells, and the endothelial cell aging model group overexpressing microRNA is endothelial cells in which endothelial cells overexpressing microRNA are exposed to hydrogen peroxide.

7. The method for screening microRNA combinations in atherosclerosis drugs according to claim 1, characterized in that: In step S5, the detection includes cell aging detection, cell viability detection, cell cycle detection and protein expression detection.

8. The method for screening microRNA combinations in atherosclerosis drugs according to claim 7, characterized in that: The cell aging detection adopts the β-galactosidase staining method, the cell viability detection adopts the CCK-8 method, the cell cycle detection adopts flow cytometer analysis, and the protein expression detection adopts WB detection.

9. A method for screening a microRNA combination in an atherosclerosis drug according to claim 7 or 8, characterized in that: The target proteins for protein expression detection include P16, P21 and phosphorylated histone H2A.X.

10. Use of the method for screening microRNA combinations in atherosclerosis drugs according to any one of claims 1 to 9 in preparing drugs for preventing or treating atherosclerosis.

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