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GW23-e1470
INSULIN AMELIORATES MIR-1-INDUCED INJURY IN H9C2 CELLS UNDER OXIDATIVE STRESS VIA AKT ACTIVATION
  1. Tao Chen,
  2. Zuyi Yuan
  1. Department of Cardiovascular Medicine, First Affiliated Hospital of Medical School, Xi'an Jiaotong University

    Abstract

    Objectives Growing evidence indicates that aberrant up-regulation of microRNA-1 (miR-1) occurs in ischaemic myocardium. In addition, insulin elicits metabolism-independent cardioprotection against cardiovascular diseases. The aim of this study was to determine whether insulin could ameliorate miR-1-induced injury in H9c2 cells under oxidative stress and to investigate the underlying mechanisms.

    Methods H9c2 cells were treated by hydrogen peroxide (H2O2). The expression level of miR-1 was detected by quantitative real-time PCR (qRT-PCR). Ad-GFP and Ad-miR-1 were transfected into H9c2 cells. MiR-1 transfected H9c2 cells were preincubated with LY294002 (10 µM), insulin (100 nM) alone or in combination for 1hr and subsequently were exposed to 200 µM H2O2. The cell viability, p-Akt/Total-Akt, and ROS production were detected by MTT, western-blot and DHE, respectively.

    Results We show that miR-1 is upregulated in H9c2 cells after treatment with H2O2, 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 H2O2 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 H2O2 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.

    Conclusions Our findings suggest that miR-1expression is sensitive to H2O2 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.

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