MicroRNA Expression Profile And Cardioprotection By MicroRNA-378 In Ischemic Cardiac Cells

Myocardial ischemia and infarction is a clinically common and frequently-occurring disease. The involvement of endogenous factors in myocardial ischemia remains barely known. MicroRNAs (miRNAs) are a class of endogenous small RNAs regulating post-transcription. Based on the base-pairing principle, miRNAs bind to complementary sequences that are usually located in the 3'-untranslated region (3'-UTR) of target mRNAs, then trigger mRNA cleavage and/or translational repression. Bioinformational analyses predicted that one miRNA can target hundreds of mRNAs, indicating the multiplicity of miRNAs function. Recent research demonstrated miRNAs play pivotal roles in the pathologic processes including myocardial ischemia. However, only a few miRNAs have been associated with myocardial ischemia. The actions of most of the miRNAs under ischemic conditions remain unknown.The present study examined the expression of miRNAs in an in vivo rat model of acute myocardial ischemia caused by coronary artery occlusion, using the methods of miRNA array analysis and quantitative real-time PCR. We identified the aberrantly-expressed miRNAs in ischemic myocardium, and picked dramatically changed miRNAs for function study in cultured rat H9c2 cardiac cells. The H9c2 cells were transfected with specific mimic or inhibitor using a liposome-based technique to induce overexpression or deficiency of the target miRNA. Cellular hypoxic injury and apoptosis were measured upon mimic or inhibitor transfection to determine whether the miRNA is protective or harmful. When the function of a miRNA was established, the potential targets of the miRNA, as predicted by bioinformatic analyses, were investigated to uncover the mediators of miRNA effects on hypoxia-induced injury and apoptosis in H9c2 cells.Methods1. The model of acute myocardial ischemia in intact ratsIn male SD rats anesthetized with ethyl carbamate, the heart was exposed by opening the chest, then the left anterior descending coronary artery (LAD) was occluded to produce myocardial ischemia. The ischemic myocardium was dissected after 4 hours. Normally perfused myocardium, which served as control, was removed from sham-operated rats that had gone through all the procedures without LAD occlusion. 2. The model of hypoxia in cultured rat H9c2 cardiac cellsStandard cultured H9c2 cells were serum deprived for 12 h for synchronization before the exposure of hypoxia. All the hypoxic and normoxic experiments were performed in cells under serum-free conditions. Hypoxia was generated by infusing nitrogen into an oxygen-regulated incubator to maintain an atmosphere of 1%O2-94%N2-5%CO2 for 24h. Cells cultured under normoxic atmosphere served as control.3. MicroRNA array analysesThe expression levels of miRNAs in normal and ischemic myocardium of rats were measured by miRNA array analyses. Briefly, total RNAs were extracted, and the quality was checked with OD260/OD280 ratio and formaldehyde denaturing agarose gel electrophoresis. Then miRNAs were isolated, labeled with fluorescent dye, hybridized with array chips, and detected for fluorescent intensities that represent miRNA levels. All the miRNA array experiments and data analyses were performed by LC Sciences, USA.4. Determination of microRNA expression levels with quantitative real-time PCRThe expression levels of miRNAs in normal and ischemic myocardium of rats, as well as normoxic and hypoxic cultures of H9c2 cells, were determined with quantitative real-time PCR. Total RNAs extracted from heart tissue or H9c2 cells were subjected to reverse transcription with a special stem-loop primer for miRNA transcription. The product of RT reaction was used for real-time PCR reactions.GAPDH was employed as an internal control, and the standard curve method was used to calculate the relative levels of miRNAs.5. Overexpression or knockdown of miR-378H9c2 cells cultured in serum-free medium were transfected with miR-378 mimic or inhibitor, with the aid of siport neofx transfection agent. The overexpression or knockdown of miR-378 was validated by real-time PCR.6. Identification of genes targeted by miR-378The potential target genes of miR-378 were bioinformatically predicted with computational tools including TargetScan, Bibiserve and Pictar, and the target genes of interest were further tested in experiments. In cultured H9c2 cells exposed to hypoxia, the expression level of the potential target genes was demined upon miR-378 mimic or inhibitor transfection, and was correlated with cellular hypoxic injury to define their roles in the actions of miR-378.Results1. Establishment of myocardial ischemia/hypoxia modelIn intact rats, myocardial ischemia was successfully induced by LAD occlusion, as evidenced by the cyanosis of ischemic myocardium, the elevation of ST-segment in ECG, the dramatic decrease of blood pressure, and the differentiation of normal and ischemic myocardium by Evans blue and TTC staining 4h after LAD ligation.In cultured H9c2 cardiac cells, hypoxia was successfully generated by 24-h incubation under 1% 02. Cellular hypoxic injury was evidenced by the decline of cell numbers, the release of LDH and the decrease of cell viability.2. MiRNA expression in myocardial ischemia/hypoxia modelMiRNA array analyses showed that multiple miRNAs are aberrantly expressed in ischemic myocardium of rats. miR-21 and miR-23a were significantly up-regulated, whereas miR-143, miR-199a-3p and miR-378 were highly down-regulated. Among the aberrantly-expressed miRNAs, miR-378 expression was significantly down-regulated by 49% in acute ischemic myocardium. The technique of real-time PCR validated most of the miRNA changes detected by miRNA array. miR-378 expression was shown to be down-regulated by 42% in real-time PCR measurements.In cultured H9c2 cardiac cells exposed to hypoxia, we also found that multiple miRNAs were aberrantly expressed. The changes of miRNA expression in hypoxic H9c2 cells were in accordance with those observed in ischemic myocardium. miR-378 expression was significantly down-regulated by 44% in this model.3. Function of miR-378 in hypoxic H9c2 cellsIn H9c2 cells, the transfection with miR-378 mimic at 50nM increased miR-378 expression by approximately 200 folds. Under the condition of hypoxia, miR-378 overexpression significantly inhibited LDH release by 10%, decreased supernatant MDA content by 27%, and increased cell viability by 15%. The percentage of apoptotic cells was decreased by 24%. In contrast, the knockdown of miR-378 expression by inhibitor transfection stimulated MDA formation and increased the apoptotic rate by 1.5 folds, though LDH release and cell viability did not change significantly. It is suggested that miR-378 is protective against hypoxic injury and apoptosis.4. Identification of genes that mediated the protection of miR-378Several miRNA target prediction algorithms were employed to search for potential genes directly regulated by miR-378. We found that caspase 3 might be a candidate target for miR-378. Western blot showed that miR-378 inhibitor resulted in an increase of caspase 3 protein levels by 1.5 fold, suggesting that decreased expression of miR-378 in ischemic/hypoxic myocytes might contribute to caspase 3 activation and subsequently trigger apoptosis. We are still doing experiments to verify that.Conclusion1. In a rat model of myocardial ischemia induced by LAD ligation, multiple miRNAs including miR-21,-23a,-143,-199a-3p were aberrantly expressed in the ischemic zone after a 4-h duration of ischemia. Notably, myocardial ischemia caused a 42% decrease in miR-378 expression. In H9c2 cells cultured under serum-free conditions, the expression of certain miRNAs was also found to be changed upon 24-h hypoxia. miR-378 expression was down-regulated by 44% in hypoxic cells compared to normoxic cells.2. Overexpression of miR-378 alleviated hypoxic injury and apoptosis in H9c2 cells, while knockdown of miR-378 expression exerted adverse effects. The results suggest that miR-378 plays a protective role in cardiac cells exposed to hypoxia.3. The cardioprotective effects of miR-378 against hypoxia are suggested to be mediated by its inhibition on caspase-3 expression.