LncRNA-AV310809在腹膜透析相关腹膜纤维化中的作用及机制研究

Peritoneal fibrosis is a common complication of long-term peritoneal dialysis, and contributes to encapsulating peritoneal sclerosis and eventually peritoneal ultrafiltration failure, which restricts the wide application of peritoneal dialysis. The prevention and treatment of peritoneal fibrosis is important to prolong peritoneal dialysis treatment. However, the mechanism of the peritoneal fibrosis is not well understood. Long noncoding RNAs (lncRNAs) are defined as RNA molecules over 200nt in length which do not encode for a protein. Recent studies indicated that lncRNAs play a critical role in regulation of diverse physiological and pathological processes. Our preliminary studies showed that there were systematic differences in the expression of lncRNAs and protein-coding RNAs between fibrosis peritoneal and normal tissue. Futhermore, the expression of lncRNA AV310809 was significantly lower (more than 5 fold) in fibrotic peritoneal, and over-expression of AV310809 can significantly promote the epithelial-mesenchymal transition (EMT) process and the synthesis of ECM proteins induced by TGF-β1. Furthermore, By Pathway and Network analysis, we found the expression of AV310809 was highly correlated with Wnt2 and Wnt5 (pearson’s correlation coefficients≧0.98), which played the key role in the Wnt signal pathway. These results indicated that lncRNA AV310809 may participates in the process of peritoneal fibrosis. Therefore, our study is to investigate the function and the regulation mechanism of lncRNA AV310809 in the process of peritoneal dialysis associtated peritoneal fibrosis by using biological informatics, real-time PCR, RIP, gene cloning and RNA interference methods in vitro/vivo researches.

腹膜纤维化是影响腹膜透析效果，导致患者退出腹透最主要的原因，目前，其发生及调控机制尚不明确，仍无有效的解决办法。在前期研究中，我们构建腹膜透析相关腹膜纤维化动物模型，并通过基因芯片进行了腹膜纤维化差异lncRNA的筛选，发现lncRNA-AV310809在纤维化的腹膜组中显著高表达，而上调AV310809的表达可促进腹膜间皮细胞EMT的发生及细胞外基质的合成。通过Pathway、Network分析，我们进一步发现AV310809与纤维化相关信号通路-Wnt通路中的重要基因Wnt2和Wnt5的表达呈高度正相关。因此，我们推测AV310809可能参与了腹膜纤维化的发生及调控。基于以上前期工作，本课题拟从动物，细胞及分子水平验证AV310809表达与腹膜纤维化的关系，并明确其对Wnt信号通路的调控作用，从而揭示腹膜透析相关腹膜纤维化发生的新机制，为临床腹膜纤维化的治疗提供新的潜在药物治疗靶点。

研究背景：通过前期lncRNA基因芯片筛选，我们寻找到了与腹膜纤维化相关的关键性长链非编码RNA（lncRNA-AV310809），共表达网络分析预测其可能通过纤维化信号通路（Wnt2/β-catenin信号通路）发挥作用。本研究进一步通过体外实验探讨该关键性lncRNA在腹膜纤维化过程中的作用及具体调控机制。.研究内容：1、体外建立腹膜间皮细胞间质转分化（EMT）模型；观察TGF- β1对wnt2/β-catenin信号通路的影响。2、体外过表达AV310809观察HMrSV5细胞形态的变化；以及对Wnt2/β-catenin信号通路的影响。3、运用MTT及划痕损伤实验观察AV310809对HMrSV5细胞增殖及迁移的影响。4、RIP验证AV310809与β-catenin的相互作用。.主要结果：1、TGF- β1可诱导HMrSV5细胞EMT发生 2、TGF- β1在诱导HMrSV5细胞发生EMT的同时上调了AV310809的表达水平；3、TGFβ-1在诱导HMrSV5细胞发生EMT的过程中Wnt2/β-catenin信号通路被活化4、过表达AV310809促进了TGF-β1 诱导的HMrSV5细胞梭形样形态改变；过表达AV310809促进了TGF-β1 诱导的HMrSV5细胞EMT的发生；过表达AV310809促进了TGF-β1 诱导的HMrSV5细胞Wnt2、β-catenin蛋白表达增加；AV310809可能抑制了HMrSV5细胞增殖及迁移修复能力。.科学意义：本项目研究发AV310809可能通过激活Wnt2/β-catenin信号通路促进TGF- β1诱导的HMrSV5细胞EMT发生而参与腹膜纤维化过程，提示AV310809可能作为临床腹膜纤维化治疗的潜在干预靶点。