Insulin promotes vascular smooth muscle cell proliferation via microRNA-208-mediated downregulation of p21.

Objective: Abnormal vascular smooth muscle cell (VSMC) proliferation is involved in the development of vascular diseases. However, the mechanisms by which insulin exerts this effect are not completely known. We hypothesize that microRNAs might be involved in insulin-induced VSMC proliferation.

Methods: VSMC proliferation was determined by [H]-thymidine incorporation; microRNAs were determined by microRNA chips and real-time PCR; and p21expression was determined by immunoblotting.

Results: In this study, we found that insulin increased VSMC proliferation and miR-208 expression. Overexpression of miR-208 increased basal and insulin-mediated VSMC proliferation. Although a miR-208 inhibitor, by itself, had no effect on VSMC proliferation, it reduced the insulin-mediated cell proliferation. Moreover, miR-208 increased the transformation of cell cycle from G0/G1 phase to the S phase. Bioinformatics analysis found that p21, a member of the cyclin-dependent kinase (CDK)-inhibitory protein family, may be the target of miR-208. Insulin decreased p21 expression in VSMCs; transfection of miR-208 also decreased p21 protein expression. In the presence of miR-208 inhibitor, the inhibitory effect of insulin on p21 expression in VSMCs was partially blocked. The interaction between miR-208 and p21 was direct. Using a luciferase reporter with entire wild-type p21 3'UTR or a mutant p21 3'UTR in HEK293 cells, we found that miR-208 decreased but neither miR-208 mimic nor the mutant p21 3'UTR had any significant effect on the luciferase activity.

Conclusion: This study indicates that miRNAs, miR-208, in particular, are involved in the insulin-induced VSMC proliferation via downregulation of its potential target, p21, a key member of CDK-inhibitory protein family.