转录因子twist启动miR-9表达促进肾小管上皮细胞转分化的分子机制研究

Epithelial to mesenchymal transition (EMT) of renal tubular epithelial cells induced by chronic hypoxia is one of the most important mechanisms of renal fibrosis. It is important for preventing renal fibrosi to study the key moleculars in this process. In our previous study, we found that Twist can promote renal EMT under hypoxia in vivo and in vitro. Twist is a new prognostic marker for renal survival in patients with CKD. However, the underling mechanism is still unclear. Thus, we found that miR-9 and CDH1 were significantly changed by up-regulating Twist in HK-2 cells through microRNA array and gene array Screening. By biological information analysis，we found there is the latent binding sites of Twist in the miR-9 gene promoter region, and there is the latent binding sites of miR-9 in the CDH1 3'UTR. From this we speculated, under chronic hypoxic condition, Twist possibly directly regulates miR-9 expression, then regulates its downstream target gene, leading to the tubular injury. We will confirm this hypothesis by in vivo and in vitro experiments based on the previous studies. This project will provide the brand-new angle of view for the exposition of mechanism of chronic hypoxic tubular injury, and the vital clue to prevent renal fibrosis.

慢性缺氧导致肾小管上皮细胞转分化（EMT）是肾纤维化的重要机制之一，研究这一过程中的关键分子对防治肾纤维化具有重要意义。我们前期研究证实Twist在缺氧性肾小管EMT中发挥重要作用，而且是预测慢性肾脏病进展的一个新分子。但Twist介导慢性缺氧性肾小管损伤的分子机制尚不清楚。我们通过microRNA及基因芯片筛选，发现Twist调控的microRNAs及靶基因中，miR-9表达显著上调，CDH1表达显著下调，生物信息学分析：miR-9基因启动子区存在Twist的潜在结合位点E-box 序列,而CDH1的3'UTR存在 miR-9的潜在结合序列，由此我们推测，慢性缺氧后Twist可能直接转录调控miR-9的表达，进而调控其下游靶基因，导致肾小管损伤。本课题将在前期研究的基础上，通过体内外实验证实这一推论。本项目的完成将为阐明慢性缺氧性肾损伤机制提供全新的视觉，为探讨新的防治手段提供重要线索。

慢性缺氧导致肾小管上皮细胞转分化是肾纤维化的重要机制之一，研究这一过程中的关键分子对防治肾纤维化具有重要意义。课题组证实Twist在缺氧性肾小管EMT中发挥重要作用，通过miRNA芯片及基因芯片筛选，发现Twist调控的microRNAs及靶基因中，miR-9表达显著上调，CDH1表达显著下调，生物信息学分析miR-9基因启动子区存在Twist的潜在结合位点E-box序列，而CDH1的3'UTR存在 miR-9的潜在结合序列。实验主要研究慢性缺氧后Twist-miR-9调控在肾小管EMT过程中发挥的作用。启动子报告基因结果显示Twist可直接启动miR-9的表达，双荧光素酶报告基因结果表明CDH1是miR-9的靶基因。收集IgAN病人血液标本中miR-9的表达水平高于正常人。正常及缺氧处理的HK2细胞，RT-qPCR结果表明miR9的表达与缺氧时间呈正相关，CDH1呈负相关，且随着缺氧处理时间的延长，纤维化相关分子CTGF、ColⅠ、ColⅢ和FN的表达升高；WB和细胞免疫荧光结果显示E-Cadherin的表达降低，α-SMA、CTGF、ColⅠ、ColⅢ和fibronectin表达升高。构建IR和UUO动物模型，随着实验时间的延长，免疫组织化学染色结果表明，纤维化相关分子α-SMA、ColⅠ、ColⅢ和fibronectin的表达升高；RT-qPCR检测肾组织中miR-9的表达升高。因此，miR-9通过调控CDH1，导致E-cadherin的表达减少，促进肾小管上皮细胞转分化，导致肾脏纤维化的发生。