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MicroRNAs (miRNAs) have been reported as key gene regulators, and they control many fundamental biological processes. Previously, we demonstrated that miR-214 had a protective effect against myocardial apoptosis and myocardial fibrosis. In this study, we sought to investigate the expression of miR-214 in L6 skeletal myoblast (SKM), the regulatory effect of miR-214 on hydrogen peroxide (H2O2) induced cell apoptosis and the underlying mechanisms of the antiapoptotic effect. MiR-214 expression was up-regulated by H2O2 in a dose and time-dependent manner in L6 SKMs. To investigate the regulatory effects of miR-214 on L6 SKM, both gain-of-function and loss-of-function approaches were applied. The results showed that miR-214 improved cell survival and inhibited cell apoptosis, and blockage of miR-214 abrogated the protective effect on cell survival and resistance to apoptosis. Phosphatase and tensin homolog (PTEN) was negatively regulated by miR-214, and PTEN inhibitor obviously reversed the effect of miR-214 blockage on enhancing cell apoptosis. In addition, miR-214 up-regulated antiapoptotic protein Bcl-2, down-regulated proapoptotic protein Bax, prevented release of cytochrome c and inhibited caspase-3 activation. In summary, H2O2-induced injury increases miR-214 expression in L6 SKM, and miR-214 contributes to the protection of L6 SKM against apoptosis via lowering PTEN and subsequently inhibiting the mitochondrial-mediated caspase-dependent apoptotic signaling pathway. [ABSTRACT FROM AUTHOR]