Effect of miR-99a on insulin-stimulated proliferation of vascular smooth muscle cells in mice and its mechanism

Objective To explore the effect of miR-99a on proliferation of insulin-stimulated vascular smooth muscle cell(VSMC) in mice and investigate its detailed mechanism. Methods The vascular smooth muscle cells derived from C57 mice were cultured by adherent method, and the cells were treated with 100 nmol/L of insulin for 48 h.Quantitative real-time PCR(qRT-PCR) was adopted to investigate the changes of miR-99a expression.Brdu assay was used to evaluate the effect of transfected miR-99a on insulininduced proliferation of VSMCs.qRT-PCR and western blot analysis were used to detect mRNA and protein expression changes of Mammalian target of rapamycin(mTOR) after miR-99a transfection in experimental group and negative control and explore the effect of miR-99a on insulin-enforced mTOR/P70S6K signaling pathway.And western blot analysis was used to detect the expression of CyclinD1 after miR-99a transfection in experimental group and negative control. Results High dose of insulin significantly decreased the expression of miR-99a.Transfection of miR-99a mimics remarkably attenuated basal and insulin-stimulated VSMCs proliferation, and downregulation insulin-activated mTOR/P70S6K signaling pathway and insulin-enforced expression of CyclinD1.In contrast, transfection of miR-99a inhibitors further enhanced insulin-activated mTOR/P70S6K signaling pathway. Conclusion MiR-99a can inhibit insulin-stimulated VSMC proliferation by downregulation of mTOR.