CN112941167A - miRNA marker for cardiovascular disease diagnosis and application thereof - Google Patents
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Abstract
A miRNA marker for cardiovascular disease diagnosis and application thereof, (1)100 mu mol/l Hcy intervenes endothelial cells to establish an apoptosis model, and cell viability staining and flow cytometry detect that the concentration Hcy can cause endothelial cell apoptosis, thereby laying a foundation for subsequent experiments; (2) after the step (1), detecting the miR-491-5p expression level by RT-qPCR; determining that miR-491-5p plays a role in endothelial cell apoptosis caused by Hcy; (3) after miR-491-5p mimics and miR-491-5p inhibitor are transfected, the expression level of miR-491-5p is detected by RT-qPCR; after the miR-491-5p mimics and miR-491-5p inhibitor are successfully transfected, a foundation is laid for reversely verifying the action of miR-491-5p on endothelial cell apoptosis caused by Hcy; (4) miR-491-5p mimics and miR-491-5p inhibitor are transfected respectively, the apoptosis condition is detected again by flow cytometry, and the action of miR-491-5p in endothelial cell apoptosis caused by Hcy is determined. The miR-491-5p plays an important role in endothelial cell injury caused by Hcy, provides a theoretical basis for detecting and diagnosing atherosclerosis at a molecular level in the future, and has potential practical value.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to a miRNA marker for cardiovascular disease diagnosis and application thereof.
Background
Cardiovascular disease (CVD) is the disease with the highest incidence and mortality worldwide. At present, the number of people suffering from cardiovascular diseases in China is 2.9 hundred million, and the death rate is the first of all the diseases and accounts for more than 40 percent of the death rate of the resident diseases. The pathogenesis of CVD is not completely clear, the risk factors of CVD mainly comprise hypertension, diabetes, anoxia, aging and the like, and the finding of a molecular machine for the occurrence and development of cardiovascular diseases is a hot spot for domestic and foreign research and attention. Most cardiovascular diseases are related to the development of Atherosclerosis (AS), in recent years, AS the living standard of people is improved, the dietary habits are changed, the working pressure is increased, the social environment is changed, and other factors influence, the incidence rate of AS is in a trend of obvious increase, and the incidence age is gradually younger, so that the early discovery, prevention and control of the occurrence and development of Atherosclerosis are one of the important targets of clinical science at present. However, early molecular diagnosis, mechanism and method of AS have not been fully elucidated. At present, most of treatment on atherosclerosis is still drug treatment, common drugs include aspirin, clopidogrel and the like, but the drug has large side effects, causes anaphylactic reaction, liver damage, kidney damage, gastrointestinal tract reaction, blood system toxicity and the like, is few in applicable people and cannot be widely popularized; carotid artery ultrasound and angiography which are used as the standard for detecting vasculopathy gold have diagnostic significance only on atherosclerosis with changed arterial structure, and the atherosclerotic lesion without changed structure is difficult to directly observe; and angiography is traumatic, and is especially not suitable for patients who are allergic to contrast agents and have poor kidney function. Therefore, the search for an early molecular diagnostic marker of the development of atherosclerosis has important significance for the prevention and diagnosis of atherosclerosis.
The AS plaque is well developed in areas of abnormal blood flow patterns, such AS vessel bends and vessel branches, and the laminar flow area is rarely developed with AS lesions. The endothelial cells are located in the innermost layer of the blood vessel, the inner side of the endothelial cells is directly contacted with the blood flow, the outer side of the endothelial cells is connected with the extracellular matrix, the endothelial cells and the extracellular matrix form an endothelial cell barrier, and the integrity of the barrier function is closely related to the maintenance of the steady state of the vascular system. The disturbance of endothelial barrier function leads to the invasion of inflammatory cells and lipid deposition in the vessel wall, which is a critical process for the development of atherosclerosis. Therefore, the complete structure and function of the endothelial cells of the arterial blood vessels can prevent various harmful substances in the blood from migrating, displacing and gathering to the lower part of the intima, thereby providing necessary guarantee for preventing diseases related to atherosclerosis. Apoptosis is a fundamental biological phenomenon of the body that plays a necessary role in the removal of unwanted or abnormal cells from multicellular organisms. Apoptosis is a key mechanism of endothelial cell injury, so that understanding of the mechanism of endothelial cell apoptosis is important for the study of cardiovascular diseases, especially atherosclerosis.
Non-coding RNA is an RNA molecule that does not translate the protein product. Only 2% of the genes in the human whole genome are protein-coding genes, the remainder being composed mostly of non-coding RNA. These sequences are currently important in pathological and physiological processes, playing an important role in a series of pathophysiological processes of lipid metabolism of atherosclerosis, endothelial cell activation, proliferation and dysfunction, inflammatory-related cell recruitment and activation, smooth muscle cell proliferation and transdifferentiation. mirnas (micrornas) are small, siRNA-like molecules encoded by the higher eukaryote genome. mirnas are well conserved in species evolution, mirnas found in plants, animals and fungi are only expressed at specific tissue and developmental stages, and miRNA tissue specificity and timing determine tissue and cell functional specificity, indicating that mirnas play multiple roles in cell growth and development. The biochemical stimulation can reduce the expression of miRNA in endothelial cells, and can also reduce the expression of adhesion molecules and the expression of genes on NF-kB channels, and inhibit the generation of AS. In addition, the expression of miRNA is also involved in regulating the activation and proliferation of endothelial cells and the senescence of endothelial cells. Therefore, the change of miRNA in endothelial cells is a common phenomenon in the generation of AS and is possibly an early biological marker of AS formation, so that the role of a key miRNA in endothelial cells is defined, and the miRNA has important potential value in the early diagnosis and prognosis evaluation of AS. In addition, in the aspect of quantitative detection, the relatively simple RT-qPCR method is adopted to detect the expression of miRNA, and the method has the advantages of simple operation, strong specificity, high sensitivity, short time and high speed. Therefore, if the expression and the effect of miRNA in endothelial cell apoptosis can be detected and verified, the method has important significance for the practical clinical application of atherosclerosis screening, and has immeasurable effect on improving the living standard and quality of people.
Disclosure of Invention
The invention aims to provide a miRNA molecular marker for cardiovascular disease diagnosis and application thereof, namely miR-491-5 p. The expression and the function of the polypeptide in the process of damaging the endothelial cells by Hcy are verified, the molecular mechanism of damaging the endothelial cells by Hcy is further understood, and a theoretical basis is provided for preventing and diagnosing cardiovascular diseases, particularly atherosclerosis.
A miRNA marker for cardiovascular disease diagnosis and application thereof, the specific technical scheme is as follows:
(1) the 100 mu mol/l Hcy intervenes endothelial cells to establish an endothelial cell apoptosis model, and cell viability staining and flow cytometry detection show that the concentration Hcy can cause endothelial cell apoptosis, thereby laying a foundation for subsequent experiments.
(2) After 100 mu mol/l Hcy intervenes in endothelial cells, RT-qPCR detects the expression level of miR-491-5 p. And determining that miR-491-5p plays a role in endothelial cell apoptosis caused by Hcy.
(3) After miR-491-5p mimics and miR-491-5p inhibitor are transfected, the expression level of miR-491-5p is detected by RT-qPCR. After the miR-491-5p mimics and miR-491-5p inhibitor are successfully transfected, a foundation is laid for reversely verifying the action of miR-491-5p on endothelial cell apoptosis caused by Hcy.
(4) miR-491-5p mimics and miR-491-5p inhibitor are transfected respectively, and the apoptosis condition is detected again by flow cytometry, so that the function of miR-491-5p in endothelial cell apoptosis caused by Hcy is determined.
Detecting the expression level of miR-491-5p in injured cells on the basis of endothelial cell apoptosis caused by Hcy, wherein the sequence of miR-491-5p is shown in SEQ ID: NO: 1.
Further, the reagents including primers were synthesized by the bio-technology company of lebr, guangzhou, and the reverse transcription kit and the fluorescent quantitative PCR kit were provided by the company Takara.
Further, the step of detecting the action of miR-491-5p comprises the following steps: firstly, establishing a cell model of endothelial cell apoptosis caused by Hcy; RT-qPCR detects the expression of miR-491-5p in endothelial cell apoptosis caused by Hcy; ③ verifying the successful transfection after miR-491-5p mimics and miR-491-5p inhibitor are transfected; and fourthly, on the basis of transfecting miR-491-5p mimics and miR-491-5p inhibitor, the change of apoptosis is detected again, and the effect of miR-491-5p in endothelial cell injury is determined.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention detects the expression level of miR-491-5p in endothelial cell apoptosis caused by Hcy.
(2) The invention verifies the effect of miR-491-5p mimics and miR-491-5p inhibitor on cell apoptosis by constructing miR-491-5p mimics and miR-491-5p inhibitor on the basis of the effects, thereby further defining the effect of miR-491-5p on endothelial cell apoptosis caused by Hcy.
(3) The miR-491-5p provided by the invention has an important role in endothelial cell injury caused by Hcy, provides a theoretical basis for detecting and diagnosing atherosclerosis at a molecular level in the future, and has important theoretical guidance and potential practical value.
(4) The method is simple and easy to implement, and is easily accepted and popularized by the masses.
Drawings
FIG. 1 shows the change of the number of red (apoptotic) cells in the group 0 (control group) and the Hcy group (experimental group) after 100. mu. mol/l of Hcy of the present invention intervenes in endothelial cells.
FIG. 2 shows the apoptosis of Hcy group 0 and Hcy group detected by flow cytometry after 100. mu. mol/l of Hcy intervenes in endothelial cells. P < 0.01 in comparison with group 0
FIG. 3 shows that the expression level of miR-491-5p is detected by RT-qPCR after 100 mu mol/l Hcy intervenes in endothelial cells. P < 0.01 in comparison with group 0
FIG. 4 is the self-validation of miR-491-5p overexpression of the invention. After miR-491-5p NC control and miR-491-5p precursor (miR-491-5p mimics) are transfected, the expression level of miR-491-5p is detected by RT-qPCR. P < 0.01 in comparison with NC group
FIG. 5 shows the self-verification of miR-491-5p inhibition in the invention. After miR-491-5p NC control and miR-491-5p inhibitor (miR-491-5p inhibitor) are transfected, the expression level of miR-491-5p is detected by RT-qPCR. P < 0.01 in comparison with NC group
FIG. 6 shows that after miR-491-5p mimics are transfected, the apoptosis change of endothelial cells is detected by flow cytometry. P < 0.01 in comparison with Hcy-NC
FIG. 7 shows that after miR-491-5p inhibitor is transfected, the change of endothelial cell apoptosis is detected by flow cytometry. P < 0.01 in comparison with Hcy-NC
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and examples.
A miRNA marker for cardiovascular disease diagnosis and application thereof, the specific technical scheme is as follows:
(1) the 100 mu mol/l Hcy intervenes endothelial cells to establish an endothelial cell apoptosis model, and cell viability staining and flow cytometry detection show that the concentration Hcy can cause endothelial cell apoptosis, thereby laying a foundation for subsequent experiments.
(2) After 100 mu mol/l Hcy intervenes in endothelial cells, RT-qPCR detects the expression level of miR-491-5 p. And determining that miR-491-5p plays a role in endothelial cell apoptosis caused by Hcy.
(3) After miR-491-5p mimics and miR-491-5p inhibitor are transfected, the expression level of miR-491-5p is detected by RT-qPCR. After the miR-491-5p mimics and miR-491-5p inhibitor are successfully transfected, a foundation is laid for reversely verifying the action of miR-491-5p on endothelial cell apoptosis caused by Hcy.
(4) miR-491-5p mimics and miR-491-5p inhibitor are transfected respectively, and the apoptosis condition is detected again by flow cytometry, so that the function of miR-491-5p in endothelial cell apoptosis caused by Hcy is determined.
The miR-491-5p plays a role in endothelial cell injury caused by Hcy, and further clarifies a molecular mechanism of endothelial cell apoptosis caused by Hcy.
1. Test object
Cell line
Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from famous Biotechnology Inc. of China.
2. Instrument and experimental reagent
2.1 Main Instrument
Clean bench (suzhou, anta); CO22Incubators (Heraeus, germany); model 5415D micro bench centrifuge (Eppendorf, Germany); ultramicrospectrophotometers (simplina, usa); gradient RCR instrument (Biometra Tone, germany); fluorescent quantitative PCR instrument (Shanghai, Feng Ling); FACSCalibur flow cytometer (USA; BD); confocal laser microscopy (Germany; ZEISS).
2.2 Primary reagents
Homocysteine (Sigma, usa); fetal bovine serum, DMEM medium (australia, Gibco); penicillin streptomycin, trypsin digestion solution, and a cell viability fluorescence assay kit (Roche, Switzerland); cDNA kit, RT-qPCR kit (Japan, Takara Co.); lipo2000 (Thermo Fisher corporation, usa); the miR-491-5p NC, miR-491-5p mimics and miR-491-5p inhibitor are constructed by Tianjin Seesque bioengineering Co.
3 method
3.1 cell culture
Culturing HUVECs in DMEM medium containing 10% fetal calf serum at 37 deg.C under 5% CO2In the incubator, when the cell density reaches about 70%, the cells are intervened for 72h by using Hcy (group 0) and Hcy (group Hcy) with final concentration of 0 mu mol/L and 100 mu mol/L, and then the cells are collected for subsequent experiments.
3.2 cell viability staining to detect apoptosis
Discarding the culture medium, washing the cells in the laser confocal dish for 3 times by PBS, adding 5ml of PBS, 5 mul of nucleic acid Dye, 25 mul of Dead Dye and 3 mul of visible Dye into a 15ml centrifuge tube, mixing the three dyes, sequentially adding 200 mul of mixed Dye into each culture dish, incubating for 30min at 37 ℃, taking out, and observing the apoptosis condition by a laser confocal microscope.
3.3 flow cytometry detection of apoptosis
Endothelial cells were digested with pancreatin without EDTA and centrifuged at 800rpm at 4 ℃ to discard the supernatant. After washing the cells 3 times with cold PBS, resuspending the cells with 400. mu.l of an Annexin V binding solution, adding 5. mu.l of an Annexin V-FITC staining solution, gently mixing, incubating at 4 ℃ for 15min in the dark, adding 5. mu.l of PI staining solution, mixing, incubating in the dark for 5min, and immediately placing in a flow cytometer to detect apoptosis changes.
3.4 fluorescent quantitative PCR (RT-qPCR) for detecting the expression of miR-491-5p
3.4.1 extraction of endothelial cell RNA
The total RNA of endothelial cells was extracted according to the kit instructions, the whole procedure was performed in a clean bench (30 min. after UV irradiation sterilization), and lysis was performed by adding lysis buffer RZ to 6-well plates, and adding 1ml RZ to each well. The solution was pipetted several times with a sampler until clear. The clear solution was left at room temperature for 5min to allow complete separation of the nucleic acid complexes. Centrifuging at 12000rpm for 5min at 4 deg.C, collecting supernatant, and transferring into a new centrifugal tube without RNase. Add 200. mu.l chloroform, cap, vortex for 15sec, and let stand at room temperature for 3 min. After centrifugation at 12000rpm for 10min at 4 ℃, the sample will be divided into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, the volume of the aqueous phase is about 500. mu.l. Transferring the water phase into a new tube, slowly adding 250 μ l of anhydrous ethanol, mixing, transferring the obtained solution into adsorption column CR3, centrifuging at 4 deg.C and 12000rpm for 1min, and removing waste liquid in the collection tube. To the adsorption column CR3 was added 500. mu.l of deproteinized solution RD, centrifuged at 12000rpm at 4 ℃ for 1min, the waste solution was discarded, and CR3 was put into a collection tube. Adding 500 μ l of rinsing solution RW into adsorption column CR3, standing at room temperature for 2min, centrifuging at 4 deg.C and 12000rpm for 1min, discarding waste liquid, repeatedly adding 500 μ l of rinsing solution RW, standing at room temperature for 2min, centrifuging at 4 deg.C and 12000rpm for 1min, discarding waste liquid, placing adsorption column CR3 into 2ml collection tube, centrifuging at 4 deg.C and 12000rpm for 2min, removing residual liquid, and standing adsorption column CR3 at room temperature for 5min for fully drying. Transferring the adsorption column CR3 into a new 1.5ml centrifuge tube, adding 60. mu.l RNase-Free ddH2O, standing at room temperature for 2min, centrifuging at 12000rpm at 4 ℃ for 2min, and measuring the RNA concentration by a spectrophotometer. To prevent RNA degradation, it was immediately stored at-80 ℃.
3.4.2 miR-491-5p reverse transcription
The step is carried out in an ultra-clean bench with an ultraviolet lamp irradiating for 30min, no RNase gun heads are used in the operation process, the following reagents in the following table 1 are sequentially and respectively added, and the whole process is operated on ice:
table 1: miR-491-5p reverse transcription system
Mixing the above systems, centrifuging instantaneously, performing reverse transcription reaction at 42 deg.C for 15min, 85 deg.C for 5sec, and 4 deg.C for infinite circulation, and storing the product at-20 deg.C for a short period and at-80 deg.C for a long period without repeated freeze thawing.
3.4.2 RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection of expression of miR-491-5p
The following systems in Table 2 were added to 200. mu.l RNase-free centrifuge tubes, respectively, in that order:
table 2: RT-qPCR reaction system
After all samples are added, vortex oscillation is carried out to mix evenly, bubbles are removed after instantaneous centrifugation, the samples are placed into a fluorescence quantitative PCR instrument for reaction, the reaction system is that the temperature is 37 ℃ for 10min, the temperature is 95 ℃ for 2sec, the temperature is 60 ℃ for 20sec, the temperature is 70 ℃ for 10sec, the reaction is carried out for 40 cycles, and U6 is used as an internal reference. Relative amount of the gene of interest is 2-△△CTAs a result, miR-491-5p was designed and synthesized by Ruibo, Guangzhou.
3.5 transfection of miR-491-5p mimics and miR-491-5p inhibitor
HUVECs cells are inoculated into a 6-well plate and cultured for 24h, the cell density reaches 70%, transfection operation is carried out strictly according to the operating instruction of a Lipofectamine 2000 kit, 5 mul of negative control NC and miR-491-5p mimics/miR-491-5p inhibitor are respectively added into 250 mul of serum-free culture medium, the mixture is gently mixed, 6 mul of Lipofectamine TM 2000 liposome is mixed with 250 mul of serum-free culture medium, and the mixture is placed for 5min at room temperature. After 5min, mixing the two solutions, and standing at room temperature for 20 min. And (3) metering the volume of each hole in the 6-hole plate to 2ml by using a serum-free culture medium, putting the 6-hole plate into a constant-temperature incubator for continuous culture, replacing the complete culture medium after 6 hours of transfection, and continuously culturing for 72 hours.
4 statistical methods
The experimental results of the research are all measured data, and are statistically analyzed by prism7.0 statistical software, and the results are shown in the specification
The expression that the comparison between two groups adopts t test, the comparison between multiple sample averages adopts One-way ANOVA test, the comparison between two groups adopts Student-Newman-Keuls test, and P less than 0.05 shows that the difference has statistical significance.
5 results
5.1 cell viability staining to detect apoptosis
The influence of Hcy on the apoptosis of endothelial cells is detected by cell viability staining, and the result is shown by using a laser confocal microscope to observe: compared with the group 0, the number of apoptotic cells (red cells) in the Hcy group was significantly increased, and the cell viability was significantly decreased.
5.2 flow cytometry detection of apoptosis
Flow cytometry analysis of endothelial cell apoptosis level changes after Hcy intervention showed that: compared with the group 0, the endothelial cell apoptosis level of the Hcy group is increased, which suggests that the Hcy can promote the endothelial cell apoptosis. The difference was statistically significant (P < 0.01) compared to group 0.
5.3 miR-491-5p expression in endothelial cells
The expression of miR-491-5P in endothelial cells of two groups is detected by RT-qPCR, and the result shows that the expression of miR-491-5P in Hcy group is reduced (P is less than 0.01) compared with the expression of miR-491-5P in group 0, and the difference has statistical significance.
5.4 transfection of miR-491-5p mimics, detection of miR-491-5p expression in endothelial cells
After miR-491-5P mimics is transfected, in order to verify the transfection efficiency, the expression of miR-491-5P in endothelial cells is detected by RT-qPCR, and the result shows that compared with an NC group, the expression of miR-491-5P in an Hcy group is increased (P is less than 0.01), and the difference is statistically significant.
5.5 transfection of miR-491-5p inhibitor, detection of miR-491-5p expression in endothelial cells
After miR-491-5P inhibitor is transfected, in order to verify the transfection efficiency, RT-qPCR detects miR-491-5P expression in endothelial cells, and the result shows that compared with an NC group, miR-491-5P expression in an Hcy group is reduced (P is less than 0.01), and the difference has statistical significance.
5.6 transfection of miR-491-5p mimics, detection of apoptosis by flow cytometry
In order to make the effect of miR-491-5P on apoptosis more clear, after miR-491-5P mimics are transfected, the apoptosis is detected by flow cytometry, and the result shows that the apoptosis rate of the mimics group is obviously reduced (P is less than 0.01) compared with the NC group on the premise of Hcy intervention, and the difference has statistical significance.
5.7 transfection of miR-491-5p inhibitor, detection of apoptosis by flow cytometry
After miR-491-5P inhibitor is transfected, apoptosis is detected by flow cytometry, and the result shows that the apoptosis rate of the inhibitor group is obviously increased (P is less than 0.01) compared with the NC group on the premise of Hcy intervention, and the difference has statistical significance.
Conclusion 6
The Hcy intervenes endothelial cells to cause endothelial cell apoptosis and damage the endothelial cells, miR-491-5p is reduced in expression, and the change of apoptosis can be changed along with the change of miR-491-5p, so that the miR-491-5p plays a role in protecting the endothelial cell apoptosis caused by the Hcy.
The invention uses Hcy to intervene endothelial cells, replicates an endothelial cell apoptosis model, observes the action of miR-491-5p in the endothelial cell injury process, provides a new theoretical basis for cardiovascular diseases such as atherosclerosis and the like, and provides a new direction for clinical prevention and treatment.
The above description is only a preferred and specific implementation of the present invention, and the scope of the present invention is not limited thereto, and any person skilled in the art can easily change the technical solution or substitute the same within the technical scope of the present invention.
Sequence listing
<110> Ningxia medical university
<120> miRNA marker for cardiovascular disease diagnosis and application thereof
<141> 2021-03-16
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> miR-491-5p
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<210> 2
<211> 22
<212> RNA
<213> miR-491-5p inhibitor
<400> 2
ccucauggaa ggguucccca cu 22
Claims (5)
1. A miRNA marker for cardiovascular disease diagnosis and application thereof, the specific technical scheme is as follows:
(1) the 100 mu mol/l Hcy intervenes endothelial cells to establish an endothelial cell apoptosis model, and cell viability staining and flow cytometry detection show that the concentration Hcy can cause endothelial cell apoptosis, thereby laying a foundation for subsequent experiments;
(2) after 100 mu mol/l Hcy intervenes endothelial cells, RT-qPCR detects miR-491-5p expression level; determining that miR-491-5p plays a role in endothelial cell apoptosis caused by Hcy;
(3) after miR-491-5p mimics and miR-491-5p inhibitor are transfected, the expression level of miR-491-5p is detected by RT-qPCR; after the miR-491-5p mimics and miR-491-5p inhibitor are successfully transfected, a foundation is laid for reversely verifying the action of miR-491-5p on endothelial cell apoptosis caused by Hcy;
(4) miR-491-5p mimics and miR-491-5p inhibitor are transfected respectively, and the apoptosis condition is detected again by flow cytometry, so that the function of miR-491-5p in endothelial cell apoptosis caused by Hcy is determined.
2. The miRNA marker for diagnosing cardiovascular diseases and the application thereof according to claim 1, wherein the step of detecting the effect of miR-491-5p in endothelial cell apoptosis caused by Hcy comprises the following steps: firstly, establishing a cell model of endothelial cell apoptosis caused by Hcy; RT-qPCR detects the expression of miR-491-5p in endothelial cell apoptosis caused by Hcy; ③ verifying the successful transfection after miR-491-5p mimics and miR-491-5p inhibitor are transfected; and fourthly, on the basis of transfecting miR-491-5p mimics and miR-491-5p inhibitor, the change of apoptosis is detected again, and the effect of miR-491-5p in endothelial cell injury is determined.
3. The miRNA marker for cardiovascular disease diagnosis and application thereof of claim 1, wherein in the establishment of the endothelial apoptosis model, the cell culture method comprises: HUVECs are cultured by a DMEM medium containing 10% fetal calf serum, and are placed in an incubator with 37 ℃ and 5% CO2, when the cell density reaches about 70%, cells are interfered for 72h by using Hcy (0 group) and Hcy (100 mu mol/L) with final concentrations, and then the cells are collected for subsequent experiments.
4. The miRNA markers for diagnosing cardiovascular diseases and the application thereof according to claim 1, wherein the apoptosis detected by cell viability staining is as follows: discarding the culture medium, washing the cells in the laser confocal dish for 3 times by PBS, adding 5ml of PBS, 5 mul of nucleic acid Dye, 25 mul of Dead Dye and 3 mul of visible Dye into a 15ml centrifuge tube, mixing the three dyes, sequentially adding 200 mul of mixed Dye into each culture dish, incubating for 30min at 37 ℃, taking out, and observing the apoptosis condition by a laser confocal microscope.
5. The miRNA marker for cardiovascular disease diagnosis and the application thereof in claim 1, wherein the detection of apoptosis by flow cytometry is as follows: digesting endothelial cells by using pancreatin without EDTA, centrifuging at 800rpm and 4 ℃, and removing supernatant; after washing the cells 3 times with cold PBS, resuspending the cells with 400. mu.l of an Annexin V binding solution, adding 5. mu.l of an Annexin V-FITC staining solution, gently mixing, incubating at 4 ℃ for 15min in the dark, adding 5. mu.l of PI staining solution, mixing, incubating in the dark for 5min, and immediately placing in a flow cytometer to detect apoptosis changes.
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| US20110144914A1 (en) * | 2009-12-09 | 2011-06-16 | Doug Harrington | Biomarker assay for diagnosis and classification of cardiovascular disease |
| CN112159842A (en) * | 2020-10-11 | 2021-01-01 | 宁夏医科大学 | Molecular marker for early warning of atherosclerosis and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110144914A1 (en) * | 2009-12-09 | 2011-06-16 | Doug Harrington | Biomarker assay for diagnosis and classification of cardiovascular disease |
| CN112159842A (en) * | 2020-10-11 | 2021-01-01 | 宁夏医科大学 | Molecular marker for early warning of atherosclerosis and application thereof |
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| Title |
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| MEI YUAN ET AL: "A functional polymorphism at miR-491-5p binding site in the 3"-UTR of MMP-9 gene confers increased risk for atherosclerotic cerebral infarction in a Chinese population", 《ATHEROSCLEROSIS》 * |
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