长链非编码RNA-xist在氧化应激诱导胰岛β细胞功能缺陷及胰岛素信号通路异常中的作用机制

Pancreatic β cell dysfunction leading to a lack of insulin secretion is the key part to type 2 diabetes mellitus. Oxidative stress is an important factor of β-cell dysfunction. Non-coding RNA involved in human body pathophysiology is closely related to many diseases. So far, little study has been examined on the relationship between non-coding RNA and diabetes. Our pilot study suggested that long non-coding RNA-xist (lncRNA-xist) is a susceptibility gene of type 2 diabetes mellitus, and co-expression with TCF7L2, a related gene of insulin signaling pathway. TCF7L2 is a necessary for maintaining insulin secretion in islet β cells. However, the mechanisms still needed to be further explored. Therefore, we propose the hypothesis that lncRNA-xist-TCF7L2 may be involved in the regulation of abnormity of insulin signaling pathway induced by oxidative in β cell, resulting in reducing insulin secretion. To test this hypothesis, we will explore the regulation of lncRNA-xist and its target gene TCF7L2 of β cell insulin signaling pathway in MIN6 cells and db/db mice model by using adenovirus vector transfection, RNA interference and molecular biology methods. We will take the new perspective of long non-coding RNA to clarify the molecular mechanism of islet β cell dysfunction in type 2 diabetes and provide new ideas and new methods for the prevention and treatment of diabetes.

胰岛β细胞功能缺陷导致胰岛素分泌缺乏是2型糖尿病发生的关键环节,氧化应激是导致β细胞功能受损的重要因素。非编码RNA广泛参与人体生理病理活动,与众多疾病密切相关。迄今对非编码RNA与糖尿病的关系仍知之甚少。我们的预实验结果提示长链非编码RNA-xist(lncRNA-xist)是2型糖尿病的易感基因,并与胰岛素信号通路相关基因TCF7L2存在共表达。TCF7L2是维持胰岛β细胞分泌胰岛素的必要条件,但其调控机制有待进一步探讨。为此我们提出假说: lncRNA-xist—TCF7L2可能参与调控氧化应激诱导的β细胞胰岛素信号通路异常,导致胰岛素分泌减少。为验证这一假说,我们将从lncRNA这个新视点,通过MIN6细胞、db/db小鼠模型，采用腺病毒转染、RNA干扰、分子生物学手段探讨lncRNA-xis对胰岛素信号通路的调控,阐明2型糖尿病β细胞功能缺陷的分子机制,为防治糖尿病提供新思路。

背景：筛选在2型糖尿病患者和正常人外周血中差异表达的lncRNA，探讨lncRNA-p3134对胰岛β细胞功能的影响及参与2型糖尿病发生发展的调控机制。.研究内容：采用lncRNA芯片构建T2DM患者外周血lncRNA表达谱，并检测lncRNA-p3134与临床指标的相关性。采用慢病毒转染技术在Min6细胞过表达lncRNA-p3134，在不同糖浓度条件下观察其表达水平，采用CCK-8法检测细胞增殖活性，流式细胞术观察细胞凋亡，GSIS测定胰岛素释放水平，并检测胰岛素相关转录因子Pdx-1、MafA、GLUT2、TCF7L2的表达水平。在db/db糖尿病小鼠转染lncRNA-p3134，行IPGTT测定胰岛素释放水平及胰岛素转录因子的表达，免疫组化观察胰腺组织胰岛素阳性细胞比例，并进一步检测lncRNA-p3134过表达对胰岛素信号通路关键蛋白的表达的影响。.结果：与正常对照组相比，2型糖尿病患者外周血lncRNA-p3134的表达水平显著升高，并与空腹血糖水平呈显著正相关，与HOMA-β指数呈显著负相关。在Min6细胞中，lncRNA-p3134的表达水平受葡萄糖浓度的动态调控，适度的高糖上调lncRNA-p3134的表达，但在糖毒性条件下，表达受到抑制。lncRNA-p3134可通过上调胰岛素关键转录因子Pdx-1、MafA、GLUT2和Tcf7l2的表达，刺激胰岛β细胞合成和分泌胰岛素。其次，在Min6细胞过表达lncRNA-p3134可抑制胰岛细胞凋亡，并对高糖诱导的胰岛细胞功能障碍起保护作用。动物实验中，在db/db糖尿病小鼠体内过表达lncRNA-p3134可显著改善小鼠糖耐量水平，增加糖负荷前后的胰岛素释放。免疫组化发现lncRNA-p3134增加胰腺组织胰岛素阳性细胞表达，进一步证实lncRNA-p3134对胰岛β细胞功能具有保护作用。lncRNA-p3134可上调胰岛素信号通路PI3K/Akt/mTOR的关键蛋白的表达。当给予PI3K通路抑制剂后， lncRNA-p3134促进胰岛素分泌的功能显著减弱。.结论：2型糖尿病特异表达的lncRNA-p3134，可能通过上调胰岛β细胞关键转录因子的表达，参与胰岛β细胞的功能保护，促进胰岛素的合成和分泌，延缓糖尿病的进展，这种保护作用可能与激活胰岛素信号通路PI3K/Akt/mTOR密切相关。