miR-486 attenuates myocardial ischaemia/reperfusion injury by inhibiting apoptosis and autophagy activation via the FOXO1/PTEN/AKT/mTOR axis.

backgroundEffective therapeutic interventions for myocardial ischaemia/reperfusion injury (MIRI) are lacking. The expression of miR-486, a cardiac-enriched microRNA, is downregulated in ischaemic myocardium; however, its functional role and underlying molecular mechanisms in MIRI remain unclear.

methodsTo overexpress miR-486 in vivo, MIRI rat models were administered miR-486 AAV. Cardiac function was assessed using echocardiography, infarct size using 2,3,5-triphenyltetrazolium chloride staining, and collagen deposition using haematoxylin and eosin and Masson's trichrome staining. In vitro, H9c2 cardiomyocytes were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) and transfected with a miR-486 mimic, miR-486 inhibitor, or respective negative controls. Dual-luciferase reporter assays were conducted in HEK-293T cells to validate FOXO1 and PTEN as direct targets of miR-486. Apoptosis- and autophagy-related protein expression was analysed using western blotting. siRNA-mediated silencing of FOXO1 or PTEN and pharmacological inhibition of AKT (MK2206) or mTOR (rapamycin) were used to establish the underlying signalling axis.

resultsAAV-mediated overexpression of miR-486 significantly improved cardiac function, reduced infarct size, and attenuated myocardial histopathological injury in MIRI rats. In H9c2 cells, the miR-486 mimic suppressed OGD/R-induced elevation of FOXO1 and PTEN protein levels, whereas the miR-486 inhibitor further augmented their expression. MiR-486 directly targeted FOXO1 and PTEN via their respective 3'-UTRs in dual-luciferase reporter assays. MiR-486 overexpression markedly reduced the Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios. Additionally, it decreased Beclin 1 and LC3-II/GAPDH and increased p62 expression, indicating concurrent suppression of apoptosis and excessive autophagy. Notably, these effects were reversed with miR-486 inhibition. Consistent findings occurred in vivo. siRNA-mediated silencing of either FOXO1 or PTEN partially reversed the pro-apoptotic and pro-autophagic effects induced by miR-486 inhibition. Under OGD/R conditions, the phosphorylation levels of AKT and mTORC1 downstream targets, including S6K and 4EBP1, were restored with miR-486 overexpression. The contribution of AKT/mTOR signalling to the protective effects mediated by the miR-486 mimic was confirmed with AKT or mTOR inhibition.

conclusionsMiR-486 attenuates MIRI by directly targeting FOXO1 and PTEN, thereby facilitating AKT/mTOR pathway activation to co-ordinately suppress cardiomyocyte apoptosis and excessive autophagy. These findings support the involvement of the miR-486/FOXO1/PTEN/AKT/mTOR axis as an important cardioprotective mechanism and identify miR-486 as a potential therapeutic target for MIRI.