Insulin ameliorates miR-1-induced injury in H9c2 cells under oxidative stress via Akt activation.

Growing evidence indicates that aberrant upregulation of microRNA-1 (miR-1) occurs in ischemic myocardium. In addition, insulin elicits metabolism-independent cardioprotection against cardiovascular diseases. The aim of this study is to determine whether insulin ameliorates miR-1-induced injury in H9c2 cells under oxidative stress and to investigate the underlying mechanisms. By quantitative real-time RT-PCR (qRT-PCR), we show that miR-1 is upregulated in H9c2 cells after treatment with hydrogen peroxide (H(2)O(2)), and this effect is both dose- and time dependent. Furthermore, expression of miR-1 decreased significantly after insulin treatment (4.5 ± 0.1 vs. 3.0 ± 0.2, p < 0.05). To determine the potential role of miR-1 in cellular injury and gene regulation, adenovirus-mediated overexpression of miR-1 was used. Overexpression of miR-1 decreased cell viability by 28 ± 2 % (n = 6, p < 0.05) and damaged Akt activation with or without H(2)O(2) treatment. To further investigate the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in miR-1-induced injury, H9c2 cells were pretreated with LY294002 (10 μM LY, a specific inhibitor of PI3K) with or without insulin (100 nM) and subjected to H(2)O(2) treatment. LY pretreatment-inhibited Akt activation, lead to increased reactive oxygen species (ROS), and further decreased cell viability induced by miR-1 (n = 6, p < 0.05, n = 9-10 cells/group, p < 0.05 and n = 6, p < 0.05) under oxidative stress. This effect was abolished by insulin. In summary, our findings suggest that miR-1 expression is sensitive to H(2)O(2) stimulation. In addition, insulin decreases miR-1 expression and induces a marked protective effect on miR-1-induced injury under oxidative stress, which may be mediated by the Akt-mediated pathway. These results provide an important, novel clue as to the mechanism of the cardiovascular action of insulin.