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.

我们先前曾报道肿瘤坏死因子α（TNFα）可以抑制视网膜细胞中胰岛素信号的转导。我们最近发现，miR15a / 16还可以降低视网膜内皮细胞（REC）和血管特异性miR15a / 16敲除小鼠中的TNFα。由于计算机程序表明miR15a可以直接结合胰岛素受体，因此我们想确定miR15a是否改变了胰岛素信号转导。我们使用基于荧光素酶的结合测定法来确定miR15a是否直接结合胰岛素受体。然后，我们使用Western印迹，ELISA和qPCR研究了miR15a是否改变了REC中以及miR15a / 16内皮细胞基因敲除小鼠和过表达小鼠中的胰岛素信号蛋白。我们还用白藜芦醇处理了一些REC，以确定白藜芦醇是否可以增加miR15a的表达，因为白藜芦醇对糖尿病视网膜具有保护作用。我们发现miR15a直接结合胰岛素受体的3'UTR。白藜芦醇处理可增加在高葡萄糖条件下生长的REC中miR15a的表达。虽然总胰岛素受体水平没有改变，但在高葡萄糖条件下生长的REC中胰岛素信号转导减少，并通过白藜芦醇治疗得以恢复。miR15a基因敲除小鼠的胰岛素受体磷酸化和Akt2水平降低，丝氨酸307（已知抑制胰岛素信号的位点）上的胰岛素受体底物1（IRS-1）磷酸化增加。相反，miR15a的过表达增加了胰岛素信号转导。综上所述，这些数据表明miR15a结合胰岛素受体并间接调节胰岛素受体的作用。