GLP-1通过microRNA信号分子网络影响胰腺的增殖和凋亡

It is confirmed that glucagon-like peptide -1(GLP-1) may have long term effects on β cells neogenesis, proliferation, and reducing β cells susceptibility to apoptosis in many animal experiments. Besides, microRNAs participate in process of formation and growth in a variety of tissues. So that we hypothesize that both GLP-1 and microRNAs can regulate gene expression in proliferation and apoptosis of pancreas, and there may be a connection between them. It is found that GLP-1 can reduce β cells apoptosis through down-regulating the expression of miR-34a in our preliminary experiments, which initially confirmed our hypothesis. Highly correlated genes and microRNAs of proliferation and apoptosis in pancreas are identified by weight gene correlation network analysis (WGCNA) and microarray. Meanwhile, improvement of endocrine function, proliferation and apoptosis in pancreas, many highly correlated genes and microRNAs are detected by applying ELISA, immunofluorescence, PCR, Western-blot and flow cytometry technology in high fat diet induced insulin-resistant animal model, Pten gene knockout(RIP-Cre Pten loxP/loxP ) mouse and pancreas cells which are treated by different concentrations of GLP-1. In a word, our study aims to exploring the signal molecular network of GLP-1, microRNAs and its downstream proteins which involved in proliferation and apoptosis of pancreas, opening a new chapter in mechanism of proliferation and apoptosis in pancreas, and pursuing new targets of protecting pancreatic β cells.

动物实验已证实胰高血糖素样肽-1（GLP-1）能促进β细胞增殖、减少β细胞凋亡，同时microRNAs也参与多种组织的形成及生长过程，因此推测二者均参与调节胰腺增生和凋亡相关基因的表达并存在相互关联，我们前期实验发现GLP-1可以通过下调miR-34a的表达而减少β细胞的凋亡，初步证实这一设想。本实验用加权基因共表达网络分析识别胰腺增生与凋亡关键节点microRNAs并用芯片观测GLP-1对其的影响；应用免疫荧光、电镜、PCR、蛋白印记及流式等技术检测GLP-1对肥胖胰岛素抵抗模型鼠、Pten基因敲除鼠及胰腺细胞内分泌功能的改善、胰腺增生与凋亡的影响、多种增生与凋亡相关基因及microRNAs表达的改变，从而研究GLP-1与microRNAs及其下游蛋白构成的参与调节胰腺增生及凋亡的信号分子网络，为探索胰腺β细胞增生与凋亡的机制开拓新思路，并希望借此寻找到保护胰腺β细胞数目的新靶点。

糖脂毒性使β细胞功能进行性衰退，动物实验已证实GLP-1能促进β细胞增殖、减少β细胞凋亡，但其抑制β细胞凋亡的机制尚不清楚。本研究发现：1、GLP-1可改善HOMA-β及HOMA-IR指数，诱导实验性胰腺炎增加RegШ,、MCP-1及IL-6的表达水平，增高miR-216a、miR-181及miR-210、miR-146a的表达水平；GLP-1下调miR-34a及miR-148的表达水平，上调其下游SIRT1及Bcl-2的表达水平。2、GLP-1下调miR-9、miR-375、miR-15a, miR-16和miR-124，上调OC-2和PDK1的表达。3、GLP-1通过下调miR-23a的表达水平，从而诱导PGC-1α的表达增高；增加UCP2的mRNA表达水平，抑制凋亡基因Bax和Bak mRNA的表达水平。4、GLP-1上调AMPK、PPARα、CPT1A、SIRT1 的表达，下调SREBP1c 基因的表达水平；GLP-1 通过调节miR-33 和miR-370 的表达，进而调控氧化应激相关基因的表达水平。5、棕榈酸刺激可以导致INS-1细胞发生内质网应激和凋亡，其发生可能与DR5相关。6、GLP-1可降低miR-27a的表达，增加ABCA1的表达，逆转的胆固醇对INS-1细胞胰岛素分泌的抑制。7、GLP-1增加INS-1细胞的胰岛素分泌，增加细胞内NMN及NAD的含量。8、DPPIV组胰岛素水平升高，血糖、胰高血糖素水平降低，胰岛GABA、Bcl-2表达增加，Bax表达降低。9、GLP-1类似物和DPP-4抑制剂可降低肝脏中chemerin表达水平，明显改善机体的胰岛素抵抗状态，并显著降低肝脏脂变的程度。10、GLP-1组及DPP-4抑制剂改善糖尿病大鼠体重、肝湿重、肝指数、肝功、血脂、胰岛素、HOMA-IR、RBP4，明显改善肝脏脂肪变性，降低RBP4表达。11、GLP-1治疗后，HOMA-β及△I30/△G30升高，HOMA-IR、BMI、HbA1c、GA、FPG、2h-PG、TG、TC均下降。12、用加权基因共表达网络分析识别胰腺增生与凋亡关键节点microRNAs并用芯片观测GLP-1对其的影响，确定GLP-1与microRNAs及其下游蛋白构成的参与调节胰腺增生及凋亡的信号分子网络。本实验揭示出GLP-1的作用机制，为其在临床上抑制凋亡提供了理论依据