Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI, United States.
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI, United States; Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI, United States.
Cell Signal. 2018 Apr;44:28-32. doi: 10.1016/j.cellsig.2018.01.016. Epub 2018 Jan 12.
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.
我们之前曾报道肿瘤坏死因子α（TNFα）可抑制视网膜细胞中的胰岛素信号转导。我们最近发现，miR15a/16 也可减少视网膜内皮细胞（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，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 blot、ELISA 和 qPCR 来研究 miR15a 是否改变了 REC 中的胰岛素信号转导蛋白，以及在 miR15a/16 内皮细胞敲除和过表达小鼠中是否改变了胰岛素信号转导蛋白。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 可直接结合胰岛素受体的 3'UTR。在高葡萄糖培养的 REC 中，白藜芦醇处理可增加 miR15a 的表达。尽管总胰岛素受体水平没有改变，但高葡萄糖培养的 REC 中胰岛素信号转导降低，用白藜芦醇处理可恢复。miR15a 敲除小鼠的胰岛素受体磷酸化和 Akt2 水平降低，而胰岛素受体底物 1（IRS-1）在丝氨酸 307 上的磷酸化增加，该位点已知可抑制胰岛素信号转导。相反，miR15a 的过表达可增加胰岛素信号转导。总之，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。它还提供了白藜芦醇对糖尿病视网膜具有保护作用的另一种机制。
