MicroRNA-181a Regulates Hypoxia/Reoxygenation-induced Cardiomyocyte Apoptosis

OBJECTIVETo investigate the effects of microRNA-181a (miR-181a) on hypoxia/reoxygenation (H/R)-induced apoptosis in H9c2 cardiomyocytes in order to understand the molecular mechanisms.BACKGROUNDMyocardial apoptosis is a significant pathophysiological event in myocardial ischemia-reperfusion injury. It is widely acknowledged that intervention of myocardial apoptosis is a very important approach to the prevention of myocardial ischemia-reperfusion injury. Bcl-2 gene which was studied in the field of apoptosis is one of the most popular topics. However, the post-transcriptional mechanism of Bcl-2 in response to hypoxia/reoxygenation-mediated oxidative stress in cardiomyocytes has not been thoroughly studied. MiRNA is a small, single-stranded RNA that is approximately 22 nucleotides (nt) long. miRNAs are widely distributed and induce both messenger RNA (mRNA) degradation and the suppression of protein translation based on sequence complementarity between the miRNA and its target.METHODSCurrent bioinformatics were performed to select miRNAs for possible interactions with Bcl-2. The expression changes of miR-181a and Bcl-2 after H/R treatment were determined by real-time PCR and Western blot, respectively.Dual luciferase assay combined with mutation and immunoblotting was used to screen and verify the bioinformatically predicted miRNAs. H9c2 cells were transfected with miR-181a mimics or miR-181a inhibitorand cultured 48h. After H/R treatment, H9c2 cells were divided into the following 4 groups:control, H/R, mimic, inhibitor groups. LDH and MDA were simultaneously measured. The changes of apoptosis in H9c2 cells were quantitatively assayed by TUNEL method. The changes in intracellular ROS were detected by DCFH-DA staining. The measurement of mitochondrial membrane Potentialwas detected byusing JC-1 probe.The expression of Bcl-2、Bax、cleaved caspase-3 in H9c2 cells were detected by Western blotting.RESULTSmiR-181a was predicted as negative regulator of Bcl-2 by three commonly utilized miR target prediction algorithms.After H/R treatment, miR-181a expressions were increased compared with normal group, whereas Bcl-2 protein expressions decreased (P<0.05). Compared with H/R group, the release of LDH and the content of MDA, the production of ROS, the apoptosis rates, Bax and cleaved caspase-3 expression were significantly decreased(P<0.05), whereasthe expression of Bcl-2 and mitochondrial membrane potential was correspond increased in inhibitor group. However, upregulation of miR-181a exacerbated these changes.CONCLUSIONSThat inhibition of miR-181a confers cardiac protection against oxidative stress-induced H9c2 cell apoptosis through the direct target Bcl-2 expression and reduces ROS generation, which is important for the maintenance of mitochondrial membrane integrity and the inhibition of mitochondrial apoptotic pathway under oxidative stress conditions.