| Background Micro RNAs(mi RNAs) have emerged as a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target m RNAs, thus regulating all kinds of pathological and physiological process. It has been reported that mi RNAs are involved in cardiomyocytes apoptosis, cardiac infarction, cardiac reconstruction, cardiac ischemic reperfusion injury, medicine precondition, etc. In our previous study, mi RNA microarray was applied to identify the different mi RNA expression in normal rats’ cardiomyocytes(n CON), heart failure rats’ cardiomyocytes(f CON) and morphine preconditioned heart failure rat’s cardiomyocytes(f MPC). We found that mi R-133b-5p was significantly down regulated in f CON, compared with n CON. Meanwhile morphine preconditioning(MPC) obviously increased mi R-133b-5p expression as we compare f MPC with f CON. Based on previous results, in this study we plan to investigate the role of mi R-133b-5p in MPC’s protective effects on cardiomyocytes and its mechanism.Methods 1. To comfirm the transfection efficiency of mi R-133b-5p inhibitor. H9c2 cells in logarithmic phase were transfected with mi R-133b-5p inhibitor(FAM labeled) when they have grown into 40-50% cell confluence. 48 hours later we observed the cells under fluorescence inverted microscope to count the the ratio of cells dots with green fluorescence/all cells and examined the decline of mi R-133b-5p levels by q RT-PCR.2. To explore the role of mi R-133b-5p in myocardial apoptosis and its mechanism H9c2 cells in logarithmic phase were divided into 3 groups: the CON group, mi R-133b-5p inhibitor group and inhibitor-NC group. Cells in CON group were normally cultured. With cells in mi R-133b-5p inhibitor and inhibitor-NC group, mi R-133b-5p inhibitor and inhibitor-NC was transfected into them respectively when they have grown into 40-50% cell confluence. 48 h later, the lactate dehydrogenase(LDH) activity in culture medium, cell apoptosis rate and apoptosis related molecule Fas levels of the cultured cells were examined. 3. To explore the role of mi R-133b-5p in MPC protecting cardiomyocytes from hypoxia-reoxygenation injury and its mechanism H9c2 cells in logarithmic phase were divided into 5 groups: the CON group, the hypoxia/reoxygenation group(H/R), the MPC group, the MPC+mi R-133b-5p inhibitor group and MPC+ inhibitor-NC group. Cells in CON group were normally cultured. The H/R group cells experienced 5 h hypoxia/1 h reoxygenation. The MPC group cells experienced 10 min 1 mmol/L morphine preconditioning before H/R. Cells in MPC+mi R-133b-5p inhibitor group and MPC+ inhibitor-NC group have been transfected with mi R-133b-5p inhibitor and inhibitor-NC for 24 h respectively before MPC. LDH level in culture medium was measured. Cell viability and apoptosis rate were determined by CCK-8 kit and flow cytometry. Apoptosis related molecule Fas levels of the cultured cells were detected by both q RT-PCR and Western Blot.Results 1. To comfirm the transfection efficiency of mi R-133b-5p inhibitor. After 48 h transfection of mi R-133b-5p inhibitor(FAM-labeled), the ratio of cells dots with green fluorescence/all cells under fluorescence inverted microscope was >80%, the mi R-133b-5p levels were aslo obviouly downregulated compared withthe normal cultured H9c2 cells(P<0.05). 2. The role of mi R-133b-5p in myocardial apoptosis and its mechanism Compared with CON, mi R-133b-5p inhibitor group cells showed obviously up-regulated mi R-133b-5p expression, higher apoptosis rate and LDH activity(P<0.05), while the mi R-133b-5p expression, apoptosis rate and LDH activity changes in inhibitor-NC group were not statistically significant. Compared with CON, mi R-133b-5p inhibitor group cells expressed higher level of Fas(P<0.05), while Fas level in inhibitor-NC group didn’t obviously changed. 3. The role of mi R-133b-5p in MPC protecting cardiomyocytes from hypoxiareoxygenation injury and its mechanismCompared with CON, decline of cell viability, increase of LDH activity and apoptosis were observed in the other H/R, MPC, MPC+ mi R-133b-5p inhibitor, MPC+ inhibitor-NC groups(P<0.05), and mi R-133b-5p expression was obviously downregulated in H/R and MPC+ inhibitor-NC(P<0.05). Compared with H/R, MPC cells survived better with higher cell viability, less LDH activity, lower apoptosis rate and decreased Fas expression(P<0.05). MPC+ mi R-133b-5p inhibitor blocked the MPC protection, seen from all the CCK-8, LDH, flow cytometry and Fas m RNA and protein results(P<0.05). While all the indexes didn’t change obviously in MPC+ inhibitor-NC group, compared with MPC.Conclusion MPC protects cardiomyocytes from hypoxia-reoxygenation injury through up-regulating mi R-133b-5p expression. The mechanism might rely on mi R-133b-5p’s regulation of Fas expression at transcription level. |
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