Effects Of MiR-590-3p On Apoptosis And Autophagy Of Cardiomyocytes After Hypoxia And Reoxygenation Injury And It’s Mechanism

Objective: To investigate the role of mi R-590-3p in regulating apoptosis and autophagy in H/R induced cardiomyocytes,and to search for its downstream target genes,and to preliminarily explore its molecular mechanism.Methods:1.Neonatal rat cardiomyocytes(NRCMS)were cultured for 1 to 3 days,and TNNC1 protein was detected by immunofluorescence to identify the purity of the cultured cardiomyocytes.2.Hypoxia 6 h after reoxygenation 6 h to establish a hypoxia/reoxygenation model,CCK-8 experiment,flow cytometry,Western blot and quantitative PCR(q RT-PCR)to explore the effects of hypoxia/reoxygenation on cell viability,apoptosis rate,the Bcl-2,Bax and cleaved caspase 3,LC3 Ⅱ / Ⅰ,p62,Beclin 1 protein and mi R-590-3p expression level.3.Transfect mi R-590-3p mimic(mimic)and its control(mimic NC)into NRCMs by transient transfection method,and check the transfection efficiency by q RT-PCR.Flow cytometry and Western blot explore the effects of up-regulation of mi R-590-3p on the apoptosis rate of NRCMs,Bcl-2,Bax,cleaved caspase-3,LC3Ⅱ/Ⅰ,Beclin-1,and p62 protein expression.4.Using bioinformatics methods,the target genes of mi R-590-3p were predicted,and the interaction between the selected target gene and mi R-590-3p was verified by the dual-luciferase reporter assay;The expression changes of target genes after upregulation of mi R-590-3p were detected by Western blot,and the expression changes of target genes after H/R were detected.5.Co-transfected mi R-590-3p mimic and pc DNA-HIF-1α to interfere with the expression levels of mi R-590-3p and HIF-1α in cardiomyocytes,followed by hypoxia/reoxygenation treatment.Subsequently,the apoptotic rate of NRCMs was detected,and the protein levels of Bcl-2,Bax,cleaved caspase-3,LC3Ⅱ/Ⅰ,Beclin-1,and p62 were also detected.Result:1.The primary cultured cardiomyocytes were observed under a fluorescence microscope for purity identification.Green fluorescence(TNNC1)is located in the cytoplasm of cardiomyocytes,and blue fluorescence(DAPI)is located in the nucleus of all cells.The purity of cultured cardiomyocytes is higher than 95%.2.Compared with the control group,the cell activity of the H/R group decreased(P<0.05),the apoptotic rate increased(P<0.01),Bax,cleaved caspase-3,LC3Ⅱ/Ⅰ,and Beclin-1 increased(all P<0.01)0.05),Bcl-2 and p62 decreased(P<0.05),and the expression of mi R-590-3p decreased significantly(P<0.01).Compared with the mimic NC group,the expression level of mi R-590-3p in the mimic group was significantly increased(P<0.01).Compared with H/R group,the apoptosis rate of mimic+H/R group was significantly reduced(P<0.05),Bcl-2 protein level increased(P<0.05),Bax and cleaved – caspase 3 protein levels decreased(all P<0.05),the protein levels of LC3Ⅱ/I and Beclin-1 decreased(all P <0.05),and the level of p62 protein increased(P <0.05).3.The results of the dual luciferase reporter assay showed that mi R-590-3p can inhibit the luciferase activity of the wild-type plasmid carrying HIF-1α 3′UTR(P<0.05),while for the plasmid luciferase with HIF-1α 3′UTR mutation sequence,the enzyme activity has no effect,confirming that mi R-590-3p can bind to the 3’UTR of HIF-1α.In addition,compared with the control group,the HIF-1α protein level in the H/R group was significantly increased(P<0.05);compared with the mimic NC group,the HIF-1αprotein level in the mimic group was significantly reduced(P<0.01).4.Western blot results showed that,compared with the pc DNA3.1 group,the expression of HIF-1α protein in the pc DNA-HIF-1α group was significantly increased(P<0.01).5.Compared with mimic+H/R group,the apoptotic rate of mimic+pc DNA-HIF-1α+H/R group was significantly increased(P<0.01),and the protein levels of Bax and cleaved caspase-3 were increased(P<0.05),Bcl-2 protein level decreased(P<0.05),LC3Ⅱ/I,Beclin-1 protein level increased(P<0.05),p62 protein level decreased(P<0.05).Conclusion:1.mi R-590-3p can reduce cardiomyocytes apoptosis and autophagy after hypoxia/reoxygenation;2.The inhibitory effect of mi R-590-3p on cardiomyocytes apoptosis and autophagy after hypoxia/reoxygenation injury is achieved by inhibiting the expression level of HIF-1α.