| Objective: Acute myocardial infarction(AMI)is one of the most critical and serious types.Therefore,the clinical treatment guidelines recommend opening the occluded blood vessels as soon as possible to restore coronary blood flow,and the restoration of oxygen supply to ischemic and hypoxic myocardium is the best therapy.However,when the revascularization of blood vessels leads to myocardial reperfusion,some patients have arrhythmia,no reflow of blood vessels,and the scope of myocardial necrosis has been enlarged.With the development and application of reperfusion methods,more and more attention has been paid to the phenomenon of reperfusion injury.At present,it has been found that some factors activate apoptosis,necrosis and other signal transduction pathways during reperfusion,which eventually lead to myocardial injury and necrosis.How to effectively prevent and alleviate myocardial ischemia-reperfusion injury has become a hot and difficult issue.With the development of gene research and bioinformatics,non coding protein RNA has been found to directly or indirectly regulate the protein encoding gene or the protein,so as to play the biological function,in which long non-coding RNA has become a research hotspot in recent years.Therefore,searching for Lnc RNAs involved in regulation and in-depth study of regulatory mechanisms have become a new entry point for prevention and treatment of myocardial ischemia-reperfusion injury.Lnc RNA GAS5(growth arrest specific 5)is considered to be a tumor suppressor,which exerts the role of tumor suppressor gene and inhibits cell proliferation,metastasis and promote apoptosis.It has also been reported to be involved in regulating the expression of apoptosis-related genes and proteins in the nervous system and many other diseases.Myocardial ischemia-reperfusion injury is closely related to apoptosis,but the role of GAS5 in it has been rarely reported.The purpose of this study is to explore the expression and mechanism of GAS5 in myocardial ischemia-reperfusion injury,and finally to reveal the role of GAS5 in myocardial ischemia-reperfusion injury,whether the targeted regulation of GAS5 gene expression can play a role in reducing myocardial ischemia-reperfusion injury and reveal the potential mechanism.Method:1.Establishment of rat myocardial ischemia-reperfusion injury model Twenty male Wistar rats were selected and randomly divided into two groups,namely the normal myocardial group and the ischemia-reperfusion injury myocardial group,with 10 rats in each group.The normal myocardium group was not treated,and the myocardial tissue was directly taken.In the ischemia-reperfusion injury group,Langendorff isolated heart perfusion system was used to prepare MIRI model of isolated heart in rats.The MIRI model was prepared by stopping 30 minutes of reperfusion and 120 minutes of perfusion.Immediately after the perfusion,the heart was removed and myocardial tissue was removed.2.Culture of H9c2 cardiomyocytes and establishment of hypoxia-reoxygenation injury model H9c2 rat myocardial cell line was purchased from Shanghai Cell Bank,Chinese Academy of Sciences.DMEM medium containing 10% fetal calf serum was cultured at 37° C and 5% CO2.When the density is about 80%,perform hypoxia treatment,replace the complete medium with a sugar-free medium,and place the cells in a hypoxic environment(37°C,95%N2,5%CO2)for 8 hours.After that,the cells were reoxygenated,the sugar-free medium was replaced with a complete medium,and the cells were cultured in a normoxic environment(37° C,5%CO2)for 2 hours.3.Cell transfection Using transient transfection method,lipo3000 was used to transfer si RNA,mi RNA mimic,mi RNA inhibitor and negative control into H9c2 cell line according to the experimental method of the instruction manual.Follow-up experiments were performed after 24-72 hours according to different experimental requirements.4.The CCK8 method was used to compare the cell viability of normal cultured cells,hypoxic-reoxygenated cells,si GAS5 or mi R-532-5p mimic/inhibitor transfected cardiomyocytes treated with hypoxia-reoxygenation.5.The extent of LDH,CKMB,and GSH enzyme activity in normal cultured cells,hypoxic-reoxygenated cells,si GAS5 or mi R-532-5p mimic/inhibitor transfected cardiomyocytes treated with hypoxia-reoxygenation.6.Hoechst staining and flow cytometry were used to determine the apoptosis of cardiomyocytes in normal cultured cells,hypoxia-reoxygenated cells,si GAS5 or mi R-532-5p mimic / inhibitor transfected cardiomyocytes treated with hypoxiareoxygenation.7.Western blot analysis was used todetect protein expression of apoptotic proteins Bcl2,Bax,Caspase3,and PTEN in normal cultured cells,hypoxia-reoxygenated cells,si GAS5 or mi R-532-5p mimic / inhibitor transfected cardiomyocytes treated with hypoxia-reoxygenation.The protein expressions of PTEN,PI3 K,and AKT proteins were measured in normal cultured cells,hypoxia-reoxygenated cells,transfected with si GAS5 or mi R-532-5p mimic / inhibitor or co-transfection cardiomyocytes treated with hypoxia-reoxygenation.8.Realtime PCR was used to detect the gene expression of GAS5 in ischemia-reperfusion injury myocardial tissue and hypoxia-reoxygenation injury cardiomyocytes;To detect the gene expression of mi R-532-5p and PTEN in hypoxia-reoxygenation injury cardiomyocytes;To detect the gene expression of GAS5,mi R-532-5p,and PTEN in hypoxia-reoxygenation treatment cardiomyocytes after transfection with si GAS5 or mi R-532-5p mimic / inhibitor.9.The luciferase reporter gene experiment was performed to detect whether GAS5 and mi R-532-5p and mi R-532-5p were bound to PTEN and verify the binding site.10.Statistical analysis: The results were shown as the mean ± standard deviation(SD).The independent sample t test was used for comparison between the two groups,and the single factor analysis of variance method was used for comparison between multiple samples.If there was a statistical difference between the groups,the LSD test was used for pairwise comparison,P < 0.05 was considered statistically significant.Results: Part 1: Compared with the control group,the gene expression of GAS5 was significantly up-regulated in rat myocardial tissue of ischemia-reperfusion and H9c2 of hypoxia reoxygenation.Based on the establishment of rat model of hypoxia reoxygenation of H9c2,functional studies were carried out.After hypoxia reoxygenation,the activity of H9c2 cardiomyocytes decreased significantly,serious cell damage appeared,and obvious apoptosis appeared.Compared with the HR+si NC group,the activity of cells in HR+si GAS5 group was restored to a certain extent,the level of myocardial enzymes was also significantly reduced,the phenomenon of apoptosis was also reduced,and the expression of apoptotic protein was also significantly changed.Part 2: Based on the model of H9c2 hypoxia and reoxygenation in rats,the regulatory mechanism of GAS5 in myocardial ischemia-reperfusion injury was studied.The bioinformatics analysis method was used to predict the ce RNA mechanism and binding targets of GAS5.PCR resultes showed that GAS5 and PTEN were highly expressed under HR conditions,while mi R-532-5p was low-expressed,which was in line with the gene expression trend of the ce RNA mechanism.The si RNA or mi RNA mimics / inhibitors were transfected into information cells,respectively,and the expression of other genes was verified by PCR.As a result,negative feedback regulation mechanism was found in GAS5,mi R-532-5p and mi R-532-5p,PTEN.Further luciferase reporter genes experiments confirmed the binding targets.At the same time,the relationship between mi RNA and MIRI was verified.Mi R-532-5p was overexpressed and silenced separately to detect changes in cell activity,degree of injury and apoptosis.The results showed that after overexpression of mi R-532-5p,the changes in cell activity,damage degree and apoptosis after HR treatment were consistent with the results of silencing GAS5,but the results after silencing mi R-532-5p were opposite.In order to further confirm the regulatory mechanism of GAS5 / mi R-532-p / PTEN / PI3K-AKT axis in HR-induced apoptosis,a rescue experiment was designed.The si GAS5 and mi R-532-5p inhibitors were separately reansfected or co-transfected into H9c2 and then subjected to HR treatment to detect the proteins expression of PTEN,PI3 K,AKT and their phosphorylated proteins.It was found that transfection of si GAS5 alone could reduce PTEN expression,and transfection of mi R-532-5p inhibitor alone could up-regulate PTEN expression.When cells were transfected with si GAS5 and mi R-532-5p inhibitor simultaneously,the trend of PTEN expression was significantly reversed.Silencing GAS5 activated the PI3 K / AKT pathway,which was inhibited by co-transduction of mi R-532-5p inhibitors.Therefore,the experimental results suggested that the silencing of GAS5 promoted the activation of the PI3 K / AKT signaling pathway,which may be caused by the inability of the latter to regulate PTEN after binding to mi R-532-5p.Conclusion: 1.GAS5 is significantly overexpressed in rat myocardial ischemia-reperfusion injury myocardial tissue and rat ischemia-reoxygenation-treated H9c2 cell line.2.Silencing GAS5 can reduce the injury of H9c2 cells induced by anoxia and reoxygenation.3.Mi R-532-5p participates in the regulation of myocardial ischemia-reperfusion,and overexpression can reduce the injury of H9c2 cells induced by hypoxia reoxygenation.4.GAS5 acts as ce RNA,adsorbs mi R-532-5p,and then regulates PTEN.Finally,it induces apoptosis through the PI3 K / AKT pathway and participates in myocardial ischemia-reperfusion injury. |
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