LncRNA-PRTS正反馈调控TGF-β/Smads通路在糖尿病肾病EMT发生中的作用及机制研究

Diabetic nephropathy (DN) is common and refractory microvascular complication of diabetic. It is known that TGF-β-mediated epithelial-mesenchymal transition of renal tubular epithelial cells plays a key role in the development of diabetic nephropathy, however, the mechanism is not clear. In our preliminary studies, we applicated the lncRNAs microarray technology to detect and screen the specific lncRNAs of diabetic nephropathy, and validated using quantitative real-time polymerase chain reaction. We found that lncRNA-PRTS was significantly upregulated in kidney tissue of diabetic nephropathy patients. Over-expression of lncRNA-PRTS can significantly promote the EMT of renal tubular epithelial cells. Application of CHIP-PCR experiments confirmed that Smad3 can bind to the promoter region of LncRNA-PRTS. We used RNA pull-down experiments to screen and found that the LncRNA-PRTS bound with the protein GADD34 that can inhibit TGF-β signaling activation. Therefore, we propose that LncRNA-PRTS can be transcripted by Smad3 and combine with GADD34 to mediate activation of TGF-β/Smads signal during diabetic nephropathy. We intend to further confirm that LncRNA-PRTS positive feedback regulation of TGF-β/Smads signal pathway, and explore the importance and significance of the incident in epithelial-mesenchymal transition of diabetic nephropathy. The study may provide new ideas and methods for the prevention and treatment of diabetic nephropathy.

糖尿病肾病（DN）是糖尿病常见且难治的微血管并发症，TGF-β介导的肾小管上皮细胞-间质转化（EMT）在DN发病中起关键作用，但机制尚不清楚。我们在前期研究中，应用LncRNAs芯片筛选到DN特异表达的LncRNA-PRTS；上调表达LncRNA-PRTS能促进肾小管上皮细胞发生EMT；应用CHIP-PCR实验证实Smad3能结合LncRNA-PRTS的启动子区域；且RNA pull-down实验筛选到LncRNA-PRTS与抑制TGF-β信号激活的蛋白质GADD34相结合。因此，我们提出DN时LncRNA-PRTS被Smad3转录调控，并且LncRNA-PRTS与GADD34结合介导激活TGF-β/Smads信号。本项目拟进一步证实LncRNA-PRTS正反馈调控TGF-β/Smads信号通路，并探讨这一事件对糖尿病肾病EMT发生的重要性和意义，为临床防治DN提供新的思路和方法。

糖尿病肾病（diabetic nephropathy, DN）是糖尿病最严重的微血管并发症之一，DN晚期出现的肾小管间质纤维化是导致DN进展为终末期肾病End-stage renal disease, ESRD)的主要病理基础之一，而TGF-β介导的上皮-间质细胞转换过程（ epithelial mesenchymal transition, EMT）被认为是肾间质纤维化发生发展的核心环节之一。越来越多证据表明lncRNAs的异常表达与DN的发生、发展密切相关，然而其参与DN的发生机制尚不明确。.本研究发现：.1..高通量lncRNAs芯片检测结果显示与MCD患者的肾组织相比，DN患者肾组织中有486个差异表达的lncRNAs，与未用TGF-β1刺激的人肾小管上皮细胞（HK-2细胞）相比，TGF-β1刺激的HK-2细胞中有126个差异表达的lncRNAs，两组芯片中有包括lncRNA ATB (lncRNA PRTS)在内的8个共同差异表达的lncRNAs，lncRNA ATB 在DN患者肾组织，高糖及TGF-β1 刺激后的HK-2细胞中表达水平增高。LncRNA ATB 主要位于HK-2细胞的胞浆。.2..LncRNA ATB促进HK-2细胞的EMT和细胞外基质蛋白合成，而下调lncRNA.ATB 的表达可以减轻高糖及TGF-β1诱导的EMT及细胞外基质蛋白合成增多。.3..LncRNA ATB 作为ceRNA 竞争性结合 miR-30c-5p 并上调 miR-30c-5p 靶基因 Snail1 的表达,进而促进 HK-2细胞的EMT 和细胞外基质蛋白的合成。