Downregulation of microRNA-199a-5p attenuates hypoxia/reoxygenation-induced cytotoxicity in cardiomyocytes by targeting the HIF-1α-GSK3β-mPTP axis

MicroRNAs (miRs) have been identified as critical regulatory molecules in myocardial ischemia/reperfusion injury; however, the exact expression profile of miR-199a-5p in reperfusion injury and the underlying pathogenic mechanisms remain unclear. In the present study, it was revealed that miR-199a-5p expression was significantly increased in the plasma of patients with acute myocardial infarction and in a H9c2 cell model of oxygen-glucose deprivation and reperfusion (OGD/R) via reverse transcription-quantitative PCR. H9c2 cells were transfected with miR-199a-5p mimic or inhibitor, or short interfering RNA (siRNA) specific to hypoxia-inducible factor-1α (HIF-1α). 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 miR-199a-5p in the OGD/R cell model significantly decreased the viability and increased the lactate dehydrogenase leakage of cells; whereas knockdown of miR-199-5p induced the opposing effects. Additionally, inhibition of miR-199-5p significantly attenuated OGD/R-induced alterations to the mitochondrial transmembrane potential (ΔΨm) and increases in the apoptosis of cells. Furthermore, the overexpression or knockdown of miR-199a-5p decreased or increased the expression of HIF-1α and phosphorylation of glycogen synthase kinase 3β (GSK3β) in OGD/R-treated H9c2 cells. Additionally, siRNA-mediated downregulation of HIF-1α decreased phosphorylated (p)-GSK3β (Ser9) levels and reversed the protective effects of miR-199a-5p inhibition on OGD/R-injured H9c2 cells. Similarly, treatment with LiCl (a specific inhibitor of p-GSK3β) also attenuated the protective effects of miR-199a-5p knockdown on OGD/R-injured H9c2 cells. Mechanistic studies revealed that HIF-1α was a target of miR-199a-5p, and that HIF-1α downregulation suppressed the expression of p-GSK3β in OGD/R-injured H9c2 cells. Furthermore, an miR-199a-5p inhibitor increased the interaction between p-GSK3β and adenine nucleotide transferase (ANT), which was decreased by OGD/R. Additionally, miR-199a-5p inhibitor reduced the OGD/R-induced interaction between ANT and cyclophilin D (Cyp-D), potentially leading to the increased mitochondrial membrane potential in inhibitor-transfected OGD/R-injured H9c2 cells. Collectively, the present study identified a novel regulatory pathway in which the upregulation of miR-199a-5p reduced the expression of HIF-1α and p-GSK3β, and potentially suppresses the interaction between p-GSK3β and ANT, thus promoting the interaction between ANT and Cyp-D and potentially inducing cytotoxicity in OGD/R-injured H9c2 cells.