RBM3 Interacts With Raptor To Regulate Autophagy And Protect Cardiomyocytes From Ischemia-reperfusion-induced Injury

Objective:To investigate the expression of RNA-binding motif protein 3(RBM3)in myocardial infarction reperfusion injury(MIRI)and the specific role and mechanism it plays in MIRI.Methods:(1)In vivo,SD rats were used to establish myocardial infarction reperfusion model,and WesternBlotting method was used to detect the expression of RBM3.In vitro,oxygen-glucose deprivation reperfusion(OGD/R)model was established by rat embryonic cardiomyocytes(H9c2),and the expression of RBM3 was detected by WesternBlotting and immunofluorescence.(2)Small interfering RNA targeting RBM3 was constructed to transfect H9c2 cells,and the expression of apoptotic proteins Bcl-2 and Bax was determined by Western Blotting.Morphological changes in the nucleus and apoptosis rate after RBM3 knockdown were observed by DAPI staining and flow cytometry,respectively.(3)The changes of autophagy-related proteins Beclin1 and LC3 under the conditions of down-regulation of RBM3 were determined using Western Blotting and immunofluorescence assays.(4)With the help of immunoprecipitation-mass spectrometry results,the proteins interacting with RBM3 were found by bioinformatics analysis,and the inter-protein relationships were verified by Western Blotting.Results:(1)The MIRI model was successfully constructed using SD rats and H9c2 cardiomyocytes,and the elevated expression level of RBM3 in this model was determined by Western Blotting.(2)H9c2 cells were successfully transfected with RBM3 specific small interference RNA,and it was found that the expression level of lactate dehydrogenase in H9c2 cells was increased;Western Blotting showed that downregulation of RBM3 promoted apoptosis-related protein expression and H9c2 cells underwent apoptosis.(3)After down-regulation of RBM3,the expression of autophagy-related proteins Beclinl and LC3 decreased,while the expression of Raptor increased;Immunofluorescence showed that LC3 protein expression intensity was reduced after RBM3 downregulation.(4)Validation by bioconductivity analysis and immunoprecipitation suggests that RBM3 may have a direct binding role to Raptor.Conclusion:The above results suggest that RBM3 may exert its protective effects on infarcted myocardium by regulating the levels of autophagy-related proteins Beclinl and LC3 through Raptor,thereby affecting the occurrence of autophagy and apoptosis.