联合microRNA测序及体外实验探究miR-15b介导PINK1/Parkin线粒体自噬通道在心力衰竭过程中的作用机制OA

Objective:To explore the mechanism of miR-15b-mediated PINK1/Parkin mitophagy pathway in the process of heart failure(HF)and to find a new target for prevention and treatment of HF.Methods:The differential expression of microRNA between patients with HF and healthy volun-teers was detected by High-throughput sequencing;HL-1 cardiomyocytes were cultured in vitro,cardiomyocyte injury model was induced by Ang II,overexpression and inhibition of miR-15b by cell transfection technique,the transfection efficiency of miR-15b and the expression level of miR-15b in each group were detected by qRT-PCR,and the targeting relationship between miR-15b and PINK1 was verified by double luciferase reporter gene experiment.Western Blot was used to detect the expression of autophagy protein Beclin-1,LC3B and mitophagy protein PINK1 and Parkin,and im-munofluorescence was used to detect the expression of PINK1 and Parkin protein in cardiomyocytes.Results:There were significant differences in the expression of microRNA between patients with HF and healthy volunteers.The level of miR-15b in patients with HF was significantly up-regulated[log2(Fold_change)>1;P<0.01].By predicting the target gene of miR-15b,it was found that PINK1 was the target,and the targeting rela-tionship between miR-15b and PINK1 was further determined by double luciferase reporter gene.In the cell experiment,compared with the blank group,the expression of miR-15b in the model group increased(P<0.05),the levels of autophagy LC3B and Beclin-1 decreased(P<0.01),and the level of mitophagy signal pathway PINK1 and Parkin decreased(P<0.01).Compared with the model group,the above trend was significantly en-hanced after transfection of miR-15b mimics(P<0.05),and could be reversed after transfection of miR-15b inhibitor(P<0.01).Conclusion:miR-15b can regulate the level of autophagy by mediating the signal pathway of PINK1/Parkin mitophagy,which may be a potential therapeutic target for HF.