miRNA15a regulates insulin signal transduction in the retinal vasculature.

We previously reported that tumor necrosis factor alpha (TNFα) could inhibit insulin signal transduction in retinal cells. We recently found that miR15a/16 also reduced TNFα in retinal endothelial cells (REC) and in vascular specific miR15a/16 knockout mice. Since in silico programs suggested that miR15a could directly bind the insulin receptor, we wanted to determine whether miR15a altered insulin signal transduction. We used a luciferase-based binding assay to determine whether miR15a directly bound the insulin receptor. We then used Western blotting, ELISA, and qPCR to investigate whether miR15a altered insulin signaling proteins in REC and in both miR15a/16 endothelial cell knockout and overexpressing mice. We also treated some REC with resveratrol to determine if resveratrol could increase miR15a expression, since resveratrol is protective to the diabetic retina. We found that miR15a directly bound the 3'UTR of the insulin receptor. Treatment with resveratrol increased miR15a expression in REC grown in high glucose. While total insulin receptor levels were not altered, insulin signal transduction was reduced in REC grown in high glucose and was restored with treatment with resveratrol. miR15a knockout mice had reduced insulin receptor phosphorylation and Akt2 levels, with increased insulin receptor substrate 1 (IRS-1) phosphorylation on serine 307, a site known to inhibit insulin signaling. In contrast, overexpression of miR15a increased insulin signal transduction. Taken together, these data suggest that miR15a binds the insulin receptor and indirectly regulates insulin receptor actions. It also offers an additional mechanism by which resveratrol is protective to the diabetic retina.