RESUMEN
MicroRNAs (miRs) have been identified as critical regulatory molecules in myocardial ischemia/reperfusion injury; however, the exact expression profile of miR199a5p in reperfusion injury and the underlying pathogenic mechanisms remain unclear. In the present study, it was revealed that miR199a5p expression was significantly increased in the plasma of patients with acute myocardial infarction and in a H9c2 cell model of oxygenglucose deprivation and reperfusion (OGD/R) via reverse transcriptionquantitative PCR. H9c2 cells were transfected with miR199a5p mimic or inhibitor, or short interfering RNA (siRNA) specific to hypoxiainducible factor1α (HIF1α). MTS, lactate dehydrogenase (LDH), TUNEL staining and flow cytometry assays were performed to determine the proliferation, LDH activity, apoptosis and mitochondrial membrane potential (ΔΨm) of H9c2 cells, respectively. The overexpression of miR199a5p in the OGD/R cell model significantly decreased the viability and increased the lactate dehydrogenase leakage of cells; whereas knockdown of miR1995p induced the opposing effects. Additionally, inhibition of miR1995p significantly attenuated OGD/Rinduced alterations to the mitochondrial transmembrane potential (ΔΨm) and increases in the apoptosis of cells. Furthermore, the overexpression or knockdown of miR199a5p decreased or increased the expression of HIF1α and phosphorylation of glycogen synthase kinase 3ß (GSK3ß) in OGD/Rtreated H9c2 cells. Additionally, siRNAmediated downregulation of HIF1α decreased phosphorylated (p)GSK3ß (Ser9) levels and reversed the protective effects of miR199a5p inhibition on OGD/Rinjured H9c2 cells. Similarly, treatment with LiCl (a specific inhibitor of pGSK3ß) also attenuated the protective effects of miR199a5p knockdown on OGD/Rinjured H9c2 cells. Mechanistic studies revealed that HIF1α was a target of miR199a5p, and that HIF1α downregulation suppressed the expression of pGSK3ß in OGD/Rinjured H9c2 cells. Furthermore, an miR199a5p inhibitor increased the interaction between pGSK3ß and adenine nucleotide transferase (ANT), which was decreased by OGD/R. Additionally, miR199a5p inhibitor reduced the OGD/Rinduced interaction between ANT and cyclophilin D (CypD), potentially leading to the increased mitochondrial membrane potential in inhibitortransfected OGD/Rinjured H9c2 cells. Collectively, the present study identified a novel regulatory pathway in which the upregulation of miR199a5p reduced the expression of HIF1α and pGSK3ß, and potentially suppresses the interaction between pGSK3ß and ANT, thus promoting the interaction between ANT and CypD and potentially inducing cytotoxicity in OGD/Rinjured H9c2 cells.
