miR-375在RAS影响胰岛β细胞胰岛素信号中的调控作用及机制研究

Recent studies have provided cumulative evidence that renin-angiotensin system (RAS) play an important role in islet damage and protection. In a recent study in our laboratory, Angiotensin Ⅱsignificantly inhibited insulin signaling pathway and supressed insulin synthesis in insulin secreting cells NIT-1. Angiotensin-（1-7）reverted the inhibitory effect of angiotensinⅡon insulin-stimulated Akt-Ser phosphorylation in NIT-1 cells. These findings suggest that RAS may mediate a modulatory role in insulin signaling. But the mechanism remains to be elucidated. We performed miRNA microarray analysis on NIT-1 cells treated by angiotensinⅡ. The miR-375 was among the most-upregulated miRNAs in NIT-1 cells, and the expression levels were validated by real-time PCR. To identify target genes that could mediate the miR-375 effects, we applied an searches through the Targets-scan program and found that peroxisome proliferative activated receptor, gamma, coactivator 1 beta (PGC-1β) ， protein kinase D 1 (PKD1) and mitogen-activated protein kinase associated protein 1 (MAPKAP1)may be target genes, based on their potential role in insulin secretion and islet cell proliferation and apoptosis. Under the basis of these results, we want to test whether the expression of miR-375 in NIT-1 cells treated with angiotensin-(1-7) is differential. Furthermore, we seek to investigate the possible involvement of miR-375 in RAS mediated islet βcells changes. To address the hypothesis that PGC-1β, PKD1 and MAPKAP1 are the potential targets of miR-375 mediated islet βcells affectd by insulin signaling, the putative 3'UTR target site downstream of a luciferase reporter gene including PGC-1β , PKD1 or MAPKAP1 will be cloned, and co-transfected into NIT-1 cells with GFP and miR-375. The study results will provide novel clues to understanding the mechanism of islet damage and protection, and lay a foundation to explore the potential strategies of islet protection aimed at miRNA.

肾素-血管紧张素系统（RAS）在胰岛损伤与修复中发挥重要作用。我们前期研究结果显示：RAS系统的重要组分血管紧张素Ⅱ（AngⅡ）可阻碍胰岛β细胞胰岛素信号传导，抑制胰岛素合成，而血管紧张素（1-7）（Ang(1-7)）可拮抗此作用。然而RAS影响胰岛素信号分子的机制尚未明确。miRNA芯片及实时定量PCR结果显示：AngⅡ作用于胰岛细胞后胰腺特异性miRNA-miR-375呈显著差异表达。经生物信息学预测，与胰岛素信号相关的PGC-1β、PKD1及MAPKAP1可能受miR-375调控。本项目拟在此基础上，进一步从动物和细胞水平证实miR-375对PGC-1β、PKD1及MAPKAP1的靶向调节作用，明确miR-375通过调控靶基因参与AngⅡ和Ang(1-7)影响胰岛素信号通路的机制。研究成果将从一个新视角揭示胰岛损伤与保护的分子机制，为开发以miRNA 为靶标的胰岛保护措施奠定基础。

肾素-血管紧张素系统（RAS）在胰岛损伤与修复中发挥重要作用。本研究从细胞水平探索了RAS组分AngII、Ang-(1-7)影响胰岛素信号分子的机制，结果显示：1.AngII增加了胰岛β细胞内胰岛素信号通路蛋白的丝氨酸磷酸化，抑制胰岛素刺激的胰岛素基因转录水平，氧化应激可能是AngII损害作用的机制之一。2.在胰岛β细胞中，Ang-(1-7)可拮抗AngⅡ对胰岛素刺激的Akt-Ser的抑制。3.Ang-(1-7)可抑制高糖诱导的胰岛β细胞氧化应激，并可抑制JNK、p38MAPK通路的活化，进而通过Mas受体介导抑制高糖诱导的胰岛β细胞凋亡。4.AngⅡ令胰岛β细胞miR-375表达上调，阻碍胰岛素信号通路。miR-375靶向调控Mapkap1介导AngⅡ影响胰岛β细胞的凋亡及胰岛素分泌，其机制可能与Mapkap1/pAkt-Ser473有关。