MicroRNA-17-3p inhibits excessive post-hypoxic autophagy and attenuates H9C2 cardiomyocytes reoxygenation injury via PTEN-Akt-mTOR signaling

Abstract

BackgroundThe microRNA (miR)-17~92 is one of the best-characterized polycistronic miRNA clusters, which encodes six individual miRNAs and contributes to various cellular and biological processes. In particular, miR-17-3p (a passenger miRNA of miR-17) has been shown to indirectly inhibit Phosphatase and tensin homolog (PTEN) and attenuate myocardial ischemia/reperfusion (I/R) injury, but the underneath mechanism is unclear. Inhibition of PTEN can upregulate Akt/mTOR signaling and therefore suppresses the excessive autophagy, which may be a mechanism that underlies myocardial I/R injury. We, thus, hypothesized that miR-17-3p may protect against hypoxia/reoxygenation (H/R) induced cell injury by inhibiting post-hypoxic excessive autophagy in H9C2 cardiomyocytes via PTEN-Akt-mTOR signaling pathway.Methods and resultsIn rat H9C2 cardiomyocytes, H/R (6 hours hypoxia followed by 6 hours reoxygenation) significantly enhanced the expression of miR-17-3p (P <0.05 vs. Control), which was concomitant by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P<0.05 vs. Control) and the levels of p62 and the ratio of LC3II/I (autophagy markers, P<0.05 vs. Control), suggesting that miR-17-3p may be involved in the pathogenesis of H/R induced autophagic cell death. To explore the potential role of miR-17-3p in H/R-induced cell injury, H9C2 cells were transfected with the miR-17-3p agomir or its negative control. The overexpression of miR-17-3p can significantly attenuate H/R-induced cell injury (reduced LDH level, P <0.05 vs. H/R) and inhibit H/R-induced excessive autophagy (decreased levels of p62 and LC3II/I, P <0.05 vs. H/R). Given the important role of PTEN-Akt-mTOR signaling in autophagy, the effects of miR-17-3p on the key events in the PTEN-Akt-mTOR signaling cascade during H/R were examined. As anticipated, miR-17-3p overexpression significantly down-regulated PTEN expression and up-regulated the levels of phosphorylated Akt1 (Thr308) and mTOR (P<0.05 vs. H/R), indicating that the miR-17-3p may protect against excessive post-hypoxic autophagic cell death in H9C2 cardiomyocytes via PTEN-Akt-mTOR signaling pathway.ConclusionUpregulating PTEN-Akt-mTOR axis and the subsequent inhibition of excessive autophagy may represent the major mechanism whereby miR-17-3p attenuates H/R injury in H9C2 cardiomyocytes.