长非编码RNA在TGF-β/Smad3介导的慢性肾损伤中的作用- - 从全转录组水平探讨其调节机制

Inflammation and fibrosis are the common manifestations of renal injury in chronic kidney disease(CKD). It is well-known that TGF-β(transforming growth factor-beta)/Smad3 signaling is a key mediator and mediates in renal inflammation and fibrosis. Although, the blockage of TGF-β signal pathway can inhibit the process of renal fibrosis, it is also reported that TGF-β1 deficient mice develop a lethal inflammation and death at 3 weeks of age. Smad3 gene-deficient mice also found has risk in autoimmune disease by impairing immunity. The complexity and distinct roles of TGF-β/Smad in CKD suggest that treatment of CKD should aim to correct the imbalance of TGF-β/Smad signaling and target the Smad3-dependent molecules related to fibrosis and inflammation, rather than to block the general effect of TGF-β. Recent evidences indicate that long non-coding RNA (lncRNA) involve in various aspects of disease and oncogenesis. In our previous study, we found that lncRNA may play a critical role in the development of CKD. In this study, we aim to use high-throughput RNA-sequencing to identify the whole-transcriptome in Smad3 wild-type and knock-out mouse models of UUO(unilateral ureteral obstruction) and anti-GBM(glomerular basement membrane) nephritis. First of all, we plan to screen Smad3-dependent lncRNAs which related to renal inflammation and fibrosis by bioinformatical analysis. Furthermore, in vitro and in vivo studies will be used to uncover the functional role of Smad3-dependent lncRNA. PCR array and whole-transcriptome analysis will used to figure out the molecules related to renal inflammation and fibrosis in the downstream of Smad3-dependent lncRNA. Moreover, clinical observation will be conduct to demonstrate the correlation between the differentially expressed lncRNA and CKD.

炎症及纤维化造成的肾损伤是各种慢性肾病进展至终末期的共同表现。研究表明，TGF-β/Smad3是导致慢性肾损伤的主要信号通路，敲除Smad3可以抑制纤维化，却会导致机体免疫低下。因此需要进一步寻找Smad3特异性靶向调控位点。近年来，长非编码RNA与疾病的关系引起了人们的重视，我们的前期研究也表明，在TGF-β/Smad3介导的肾损伤过程中，长非编码RNA可能发挥了重要的作用。本课题旨在借助高通量测序，在慢性肾病模型中寻找与炎症及纤维化相关的转录组信息，筛选出Smad3依赖的lncRNA。然后，通过体内、外实验了解Smad3依赖的lncRNA在炎症及纤维化中的作用，并结合转录组分析寻找其下游调控基因。进一步在CKD患者肾组织中检测与肾损伤相关的lncRNA及其下游信号分子的表达，分析这些转录本的表达变化与CKD发生发展的相关性，找到TGF-β/Smad3介导肾脏损伤的调控新靶点。

炎症及纤维化造成的肾损伤是各种慢性肾脏病（chronic kidney disease, CKD）进展至终末期的共同表现。TGF-β/Smad3是导致慢性肾损伤的主要信号通路，敲除Smad3可抑制纤维化，却会导致机体免疫低下。因此需进一步寻找Smad3特异性靶向调控位点。通过本课题研究发现，在TGF-β/Smad3介导的CKD模型中，大量长非编码RNA(long non-coding RNA, lncRNA)发生了差异表达，提示它们可能在CKD的发生发展中发挥了重要的作用。本课题组挑选在CKD模型中上调倍数最高的lncRNA Arid2-IR进行了体内外功能研究。Arid2-IR表达于肾小管上皮细胞，在正常肾小管中仅有微弱的表达，但在UUO(unilateral ureteral obstruction)模型小鼠肾小管中表达显著增强。进一步证实Smad3能够作用于其启动子区域。体外下调Arid2-IR对TGF-β1诱导的细胞外基质增加并无显著作用，但能显著抑制IL-1β诱导的炎症反应，包括NF-κB的磷酸化及炎症因子IL-1β、MCP-1、TNF-α的表达。在体内，我们采用超声微泡法在小鼠肾脏中过表达Arid2-IR shRNA质粒抑制小鼠肾脏Arid2-IR的表达水平。结果表明，下调Arid2-IR表达对UUO诱导的肾脏纤维化并无显著作用，但能显著抑制炎症，包括抑制炎症细胞浸润，炎症因子的表达及炎症转录因子NF-κB磷酸化。为了进一步找到在CKD的发生过程中Arid2-IR的下游作用靶点。我们在小鼠肾小管上皮细胞中下调Arid2-IR水平并进行RNA测序,筛选下游受到Arid2-IR的调控的分子。结果显示下调Arid2-IR能够显著增加Cebpb、Nlrc5等炎症相关分子的RNA及蛋白水平。进一步利用RNA pull-down实验及质谱分析发现Arid2-IR在IL-1β诱导的炎症反应过程中可能通过与细胞骨架蛋白filamin A的相互作用从而影响mRNA的转录及选择性剪切。通过本课题的研究，我们应用新一代高通量RNA测序技术筛选在CKD过程中TGF-β/Smad3依赖的lncRNA，并结合全转录组分析，寻找新的TGF-β/Smad3介导的肾脏损伤的特异性信号通路，为日后临床防治CKD提供更加完善的科学依据和新的靶点。