The Mechanism Of MiR-92a In Attenuating Myocardial Ischemia/Reperfusion Injuryby Targeting Smad7

Background and purpose: Coronary heart disease (CHD) is the leading cause ofdeath and disability worldwide. According to the WHO,7,254,000deaths worldwide(12.8%of all deaths) resulted from CHD in2008. The effects of CHD are usuallyattributable to the detrimental effects of myocardial ischemia/reperfusion (I/R) injury.MicroRNAs (miRNAs) are a class of small (18–25nucleotides), endogenous,non-coding RNAs that silence protein expression by interacting with the3’-untranslated regions (3’UTRs) of target mRNAs. Accumulating evidence hasdemonstrated that miRNAs regulate major cellular processes involved in myocardialI/R injury, including cell proliferation, apoptosis and angiogenesis. We presume thatthe abnormal expression of miR-92a may be closely related with the development ofmyocardial I/R injury. The aim of this study is to elucidate the mechanism of reglutionof miR-92a on Smad7at post-transrciptional level and to better understand themolecular mechanism of myocardial I/R injury and development at the miRNAlevel.Methods: miR-92a targeting Smad7were predicted by bioinformatics and validatedby luciferase reporter analyses, Western blot, and quantitative real-time PCR. Thespecific miR-92a mimics or inhibitors were transfected into cardiomyocyte cells toup-regulate or down-regulate the expression levels of miR-92a, or miR-92a inhibitorand siRNA Smad7were cotransfected into cardiomyocyte cells to down-regulate theexpression levels of Smad7mRNA and protein. Then the cells underwent I/R injury.After that, cell viability, injury, and apoptosis were investigated in vitro. Finally, theexpression levels of Smad7mRNA were determined by qRT-PCR, and the proteinlevels of SMAD7and NF-B p65were determined by Western blot respectively. Results: Based on bioinformatic algorithms, miR-92a was predicted to directly target3’-untranslated region (3’UTR) of Smad7mRNA, which was verified by dualluciferase reporter gene assay. Translation of SMAD7protein was inhibited bymiR-92a at the post-transcriptional level. Compared with the I/R group, inhibition ofmiR-92a could increase cell viability, decrease cell injury, and attenuate cell apoptosis,however these effects were counteracted after co-transfection with siRNA-Smad7.Western blotting analysis was employed to quantify the protein levels of SMAD7andNF-B p65. Transfection with miR-92a inhibitor significantly increased the levels ofSMAD7protein and decreased the levels of nuclear NF-B p65protein comparedwith I/R group, which could be counteracted by co-transfection with siRNA-Smad7.However, transfection with miR-92a mimic had no effect on the protein levels ofSMAD7and nuclear NF-B p65.Conclusions: Based on the above results, inhibition of miR-92a promotes thesynthesis of SMAD7and inhibits nuclear NF-B p65proteins in myocardial I/Rinjury, which play important roles in the anti-apoptosis signaling pathway.