Effect And Mechanism Of MiR-146a-5p On Intermittent Hypoxia-Induced Injury In H9c2 Cells

Objective:1.To study the expression of mi R-146a-5p under intermittent hypoxia(IH)and the effect of IH on H9c2 cells.2.To study the effect of mi R-146a-5p in IH-induced myocardial injury.3.To study the interaction between mi R-146a-5p and XIAP,and to expore potential mechanism of XIAP in IH-induced myocardial damage.Methods:1.We exposed H9c2 cells to IH condition;the expression levels of mi R-146a-5p were detected by RT-q PCR.2.Cell viability was measured by Cell Counting Kit-8(CCK-8)and Ed U(5-ethynyl-2’-deoxyuridine)-594 assay.Cell apoptosis was assessed via flow cytometry with Annexin-V/PI staining,Hoechst 33342/propidium iodide(PI)staining.And the expressions of apoptosis-associated proteins were tested by western blotting.3.The target genes of mi R-146a-5p were predicted and verified by bioinformatics analysis and luciferase assays,respectively.4.The effect and potential mechanism of XIAP in IH-induced myocardial injury were assessed by rescue experiment.Results:1.The result of RT-q PCR showed thay mi R-146a-5p expression level was increased in H9c2 cells after IH(p<0.001).2.The CCK-8 assay showed that the cardiomyocyte viability of the IH group significantly decreased,and the Ed U-594 test demonstrated that the ratio of Ed U proliferation positive cells in the IH group was significantly reduced,indicating that the growth viability of H9c2 cells under IH conditions was significantly reduced.In addition,The flow cytometry-FITC/PI double staining method showed that IH significantly increased the apoptosis rate of cardiomyocytes,the Hoechst 33342/PI fluorescence test showed a significant increase in the proportion of cardiomyocytes in the IH group,and Western blot found that Bax and Caspase-3 protein expression in the IH group was significantly up-regulated,while Bcl-2 protein expression was significantly decreased,suggesting that the level of H9c2 cells increased significantly after IH exposure.3.The CCK-8 assay and the Ed U-594 test showed that the myocardial proliferation level of the IH group transfected with the mi R-146a-5p inhibitor was significantly increased when compared to IH group transfected with inhibitor NC.Flow cytometry-FITC / PI double staining method,Hoechst 33342 / PI fluorescence test and Western blot found that the inhibition of mi R-146a-5p expression in IH group significantly reduced apoptosis of H9c2 cells.4.Bioinformatics showed that the seed sequence of mi R-146a-5p is completely complementary to the 3’UTR site of X-linked apoptosis inhibitory protein(XIAP),suggesting that XIAP is likely to be a target gene of mi R-146a-5p.Dual-luciferase reporter assay found that luciferase activity was markedly decreased in cardiomyocytes cotransfected with mi R-146a-5p mimics and WT-XIAP compared to that of cotransfection with mimics-NC and WT-XIAP,while the luciferase activity of cardiomyocytes cotransfected with mi R-146a-5p mimics and Mut-XIAP was not significantly different from that of cotransfection with mimics-NC and Mut-XIAP,confirming that XIAP was the direct downstream target of mi R-146a-5p.5.In rescue experiment,H9c2 cells were transfected with si-XIAP,mi R-146a-5p inhibitor,or corresponding,the effects of mi R-146a-5p silence on cell viability,cell apoptotic rate,and expression levels of apoptosis-related proteins were all reversed through XIAP knockdown compared to the negative control group under IH condition negative control.Conclusion:1.The expression of mi R-146a-5p was increased in H9c2 cells under IH condition and IH could lead to myocardial damage.2.mi R-146a-5p inhibition could protect H9c2 cells from IH-induced injury.3.mi R-146a-5p mediates IH-induced cell injury by regulating XIAP expression.