转录因子Twist调控lncRNAp21及其靶分子PEPD参与慢性缺氧导致的肾脏纤维化

Chronic hypoxia-induced renal tubular injury is one of the important cause of renal fibrosis, the study of key molecules in this process is important for the prevention and treatment of renal fibrosis. Our early study confirmed that Twist plays an important role in hypoxic renal tubular injury. However, the underlying mechanism is still unclear. We found that lincRNA-p21 of Twist-regulated lncRNA expression was significantly increased by lncRNA chip screening; and lincRNA-p21 gene promoter region exists E-box sequence, which could be bind to Twist, by bioinformatics analysis. At the same time, RNA-ChIP and protein spectra showed that lincRNA-p21 binds closely to PEPD, which related to collagen metabolism. RT-qPCR results showed that the expression of Twist, lincRNA-p21, ColⅣ was positively correlated with hypoxia time in hypoxia-induced HK2 cells. From this we speculated, under chronic hypoxic condition, Twist possibly directly regulates lincRNA-p21 expression, then regulates its downstream target gene PEPD, leading to the tubular injury, collagen deposition and renal fibrosis. This study will further confirm the role of this pathway in the process of renal fibrosis based on the previous studies, and provide the basis for finding new treatment strategies.

慢性缺氧诱导的肾小管上皮细胞损伤是肾纤维化的起始阶段和可逆环节，探索此过程的关键调控分子可预防及逆转肾纤维化。课题组前期证实Twist在缺氧性肾小管损伤过程中发挥重要作用，其具体调控机制尚需深入研究。前期通过lncRNA芯片筛选，发现Twist调控的lncRNA分子中lincRNA-p21表达显著上调，生物信息学分析lincRNA-p21基因启动子区存在Twist结合位点E-box序列。RNA-ChIP及蛋白质谱结果显示，lincRNA-p21与胶原代谢相关分子PEPD紧密结合。缺氧处理的HK2细胞，RT-qPCR结果表明Twist、lincRNA-p21、ColⅣ等的表达与缺氧时间呈正相关。推测慢性缺氧后Twist可能直接调控lincRNA-p21，进而调控PEPD使胶原沉积，导致肾纤维化发生。本课题将在前期研究基础上，进一步验证此通路在肾纤维化过程中的作用，为寻找新的治疗策略提供依据。

低氧诱导的肾小管上皮细胞损伤最终导致肾脏纤维化的发生，这一过程中众多分子以及信号通路参与调控，我们课题组采用转录因子活性谱芯片（OATFA）方法对慢性缺氧性肾小管上皮细胞(HK2) 和慢性缺氧性肾损伤患者肾组织进行高通量筛选，发现Twist是其中活性差异显著的转录因子之一，研究证实：（1） Twist在慢性缺氧导致的肾小管上皮细胞损伤中发挥着重要作用。（2）Twist在慢性缺氧性肾病患者肾组织中的表达与肾功能显著负相关，它可以作为慢性肾脏病进展的分子标志。（3）Twist可在其他器官，如腹膜纤维化过程中调控细胞损伤和纤维化的发生。同时通过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。我们采用lncRNA芯片技术分析，对转染Twist慢病毒正义表达载体的HK2细胞和亲本细胞进行了高通量筛选，分别发现了234个差异表达的lncRNA分子，我们对筛选得到的10个差异变化明显的lncRNA分子进行验证分析，发现lincRNA-p21在慢性缺氧后肾小管上皮细胞以及慢性缺氧性肾损伤患者肾组织中表达明显升高。同时，进行生物信息学分析发现，在lincRNA-p21基因启动子区存在Twist的潜在结合位点E-box 序列(CANNTG)。这提示，Twist有可能直接转录调控lincRNA-p21的表达。PEPD基因与胶原代谢紧密相关，在细胞表型转换中发挥着重要的作用。更让我们感兴趣的是，经生物信息学分析发现：PEPD基因的3’UTR区有lincRNA-p21的潜在结合序列。我们的结果提示：在慢性缺氧状态下，Twist有可能直接转录调控lincRNA-p21的表达，而lincRNA-p21的表达进一步负调控其靶基因PEPD，从而促进细胞外基质的沉积，最终导致肾脏纤维化。 本课题将在前期研究的基础上，利用启动子报告基因、ChIP实验等证实lincRNA-p21受Twist调控的上游分子通路；并通过干预lincRNA-p21的表达水平，检测肾小管上皮细胞损伤情况和肾间质纤维化的发生，采用RNA-ChIP、蛋白质谱分析、Western blot和报告基因技术验证受lincRNA-p