The Role Of MicroRNA-135a In The Injury Induced By Hydrogen Peroxide In Rat Cardiomyoblast Cells

Ischemic heart disease is one of the number1cause of morbidityand mortality in the world due to aging, obesity, diabetes, and othercomorbid diseases. The potent therapeutic stategries for ischemic heartdisease is to reduce oxygen consumption, inhibit cardiomyocyteapoptosis, increase coronary flow, and induce revascularization.Reactive oxygen species (ROS)-induced cardiac cell injury plays acritical role in the pathogenesis of ischemic heart diseases. Various genes,transcription factors, and the oxygen-sensing signaling pathways areresponsible for ROS-mediated cardiac cell injury.Apoptosis is a type of programmed cell death and is highlymodulated by both intrinsic and extrinsic apoptotic factors. The extrinsicpathway is activated through transmembrane death receptors, includingFas, tumor necrosis factor (TNF) and TNF-related apoptosis-inducingligand, whereas the intrinsic pathway is mainly regulated by the Bcl-2superfamily, including Bax, Bak, Bcl-2and Bcl-XL. Bcl-2is a keyintrinsic regulator of the apoptotic cascade, and it can block apoptosis bypreventing mitochondrial permeabilization. The studies havedemonstrated that the selective inhibition of Bcl-2promotes apoptosisduring various pro-apoptotic attacks. Furthermore, accumulatingevidence from both in vivo and in vitro studies has demonstrated thatBcl-2plays important roles in the development of cardiac cell injury.However, the detailed mechanism by which Bcl-2is regulated duringdisease progression requires further investigation. MicroRNAs (miRNAs), small noncoding RNAs, play critical rolesin gene expression via inhibition of translation or degradation of targetmolecules, and are involved in the regulation of almost all major cellularfunctions, including cell differentiation, growth, mobility and death.Recent studies showed that miRNAs regulate multiple biologicalfunctions in the healthy and diseased heart, thereby contributing todisease development. An increasing number of reports confirm thatmiRNAs can function as either pro-apoptotic or anti-apoptotic factors.However, considering the critical role of Bcl-2during the developmentof cardiac cell injury, whether a miRNA might regulate Bcl-2andthereby affect disease progression remains unclear.Methods(1) H9c2cells viability was determined by MTT assays.(2) Mitochondrial membrane potential was measured with JC-1staining and flow cytometry analysis. The cell apoptosis rate wasdetected with AnnexinV-PI staining by flow cytometry.(3) The expression of microRNA-135a RNA were detected byqRT-PCR.(4) H9c2cells were transfected with microRNA-135a mimics orinhibitor, and the cell apoptosis rate was detected with AnnexinV-PIstaining by flow cytometry. Western Blot analysis to determine theexpression level of Bcl-2, Bax, Caspase-3and Cleaved caspase-3proteins.(5) H9c2cells were transfected with si-Bcl-2.The expression ofBcl-2mRNA was detected by qRT-PCR. The expression of Bcl-2proteinwas detected by Western blot. H9c2cells were transfected with si-Bcl-2and microRNA-135a inhibitor, the cell viability was determined by MTTassay, the cell apoptosis rate was detected with AnnexinV-PI staining by flow cytometry.Results(1) Certain doses of H2O2significantly inhibits H9c2cells growth at24h, and1mM H2O2for different time also inhibits H9c2cells growth.(2) H2O2treatment decreased mitochondrial membrane potentialand induces cell apoptosis.(3) H2O2treatment increased the expression of microRNA-135aRNA.(4) H9c2cells were transfected with microRNA-135a mimics andup-regulated the expression of microRNA-135a RNA. Cells transfectedwith microRNA-135a mimics significantly inhibits H9c2cells growthand enhanced the expression of Bax and Cleaved caspase-3proteinswhich indicate H2O2could efficiently induce intracellular apoptosisthrough mitochondrial pathway in H9c2cells. MicroRNA-135ainhibited the expression of Bcl-2protein but not Bcl-2mRNA.(5) In H9c2cells transfected with microRNA-135a inhibitordown-regulated the expression of microRNA-135a RNA. Cellstransfected with microRNA-135a inhibitor significantly increased H9c2cells growth treated with H2O2and inhibited the levels of Bax andCleaved caspase-3proteins.(6) In H9c2cells transfected with si-Bcl-2, the expression Bcl-2protein and mRNA were both down-regulated. H9c2cells transfectedwith si-Bcl-2and microRNA-135a at the same time, the cell viabilitywere not changed. These results indicated that Bcl-2is the target ofmicroRNA-135a.ConclusionsIn this study, we hypothesize that miRNA may play a significantrole in regulating apoptosis after in rat cardiomyoblast cells. We previously demonstrated for the first time that delivery of miR-135athrough a nonviral minicircle vector in the ischemic heart can improveheart function by promoting angiogenesis and inhibiting apoptosis.In the present study, we have demonstrated that the H2O2-inducedin vitro model of H2O2involves the miR-135a/Bcl-2signaling pathway.miR-135a was strongly upregulated in H9c2cells after H2O2treatment,accompanied by the downregulation of Bcl-2. Suppression of miR-135aby a specific inhibitor directly enhanced the expression of Bcl-2andameliorated the induction of apoptosis by H2O2. Furthermore, we knockeddown the expression of Bcl-2in H9c2cells under H2O2-treated conditions.Interestingly, we found that inhibition of miR-135a did not attenuateapoptosis in cells with Bcl-2knockdown, demonstrating that miR-135aregulates apoptosis at least partially through the regulation of Bcl-2. Tothe best of our knowledge, this is the first study examining the role ofmiRNAs in mediating apoptosis via Bcl-2in an H2O2-induced model.Previous studies have indicated that Bcl-2overexpression renders manycell types resistance to diverse apoptotic stimuli. Conversely, we andothers have demonstrated that Bcl-2-deficient cells are more susceptibleto apoptotic induction. Combined with the suppression of Bcl-2afterH2O2treatment, it is conceivable that Bcl-2is involved in H2O2-inducedapoptosis. In summary, our results demonstrate a mechanism by whichmiR-135a regulates H2O2-induced apoptosis in H9c2cells via thetargeting Bcl-2. These findings may pave a novel therapeutic avenue forIschemic heart disease induced by H2O2.