LncRNA-PFDN调控Snai1表达在糖尿病肾病发病中的作用及机制研究

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes. Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells plays an important role in renal fibrosis of DN. However, the mechanism is not fully elucidated. In our preliminary study, we performed an lncRNA microarray analysis and identified lncRNA-promoting fibrosis of diabetic nephropathy (LncRNA-PFDN) as a differentially expressed lncRNA in DN. Moreover, over-expression of LncRNA-PFDN promoted the EMT of renal tubular epithelial cells. Furthermore, by using bioinformatics methods combined with luciferase reporter assays, we demonstrated that LncRNA-PFDN can bind the miR-30a. Published study of our lab has reported that the miR-30a can inhibit EMT by targeting Snai1, which is an important transcript factor of EMT. Therefore, we proposed that LncRNA-PFDN upregulated Snai1 by competitively binding the miR-30a as a competing endogenous RNA to sponge miR-30a from targeting Snai1 and then induced EMT of renal tubular epithelial cells. This study intends to further explore the role of LncRNA-PFDN in renal fibrosis of DN, which may provide a potential novel and specific LncRNA therapy for DN.

糖尿病肾病（DN）是糖尿病危害严重的并发症。肾小管上皮细胞-间质转化（EMT）是DN肾脏纤维化的关键环节，但其机制尚未完全清楚。课题组的前期研究，应用LncRNAs芯片筛选到DN相关差异表达的LncRNA-PFDN；上调表达LncRNA-PFDN能促进肾小管上皮细胞发生EMT；应用生物信息学分析和荧光素酶报告基因实验发现LncRNA-PFDN能与miR-30a结合；本单位已发表研究发现miR-30a通过靶向调控EMT发生的关键转录因子Snai1抑制EMT发生。因此，我们提出LncRNA-PFDN作为竞争性内源RNA竞争结合miR-30a，抑制miR-30a对Snai1的靶向调控，从而上调Snai1的表达，促进肾小管上皮细胞发生EMT。本项目拟进一步阐明这一事件并探讨其对DN肾脏纤维化的重要性及意义，为临床防治DN提供新策略。

糖尿病肾病（DN）是严重影响糖尿病预后的并发症，目前尚缺乏有效的治疗方法，是我国终末期肾脏病的第二位病因，且呈现快速上升趋势。肾小管上皮细胞的炎症反应是DN肾脏小管间质纤维化的关键环节之一，但其机制尚未完全清楚。本研究应用lncRNAs芯片筛选到DN相关差异表达的lncRNA；应用原位杂交及QPCR方法在2型糖尿病db/db小鼠肾组织及高糖培养的肾小管上皮细胞中验证了lnc-PFDN呈上调表达，提示lnc-PFDN可能在DN中具有潜在作用。进一步通过细胞及分子生物学实验验证和探索了lnc-PFDN在DN中的作用及机制。本研究发现：（1）高通量芯片结果提示与db/m小鼠肾组织相比，16周龄db/db的DN小鼠肾组织中有686个差异表达的lncRNAs，其中lnc-PFDN是上调表达的新报道lncRNA。Lnc-PFDN在db/db的DN小鼠肾组织中的表达明显增高。Lnc-PFDN在高糖刺激小鼠肾小管上皮细胞后表达明显增高。原位杂交结果提示lnc-PFDN位于小鼠肾小管上皮细胞的胞浆。（2）细胞学体外实验表明，上调lnc-PFDN能促进肾小管上皮细胞发生炎症和纤维化；而应用siRNA下调lnc-PFDN能改善肾小管上皮细胞的炎症和纤维化。（3）应用生物信息学分析lnc-PFDN与miR-30a存在结合位点。荧光素酶报告基因实验发现lnc-PFDN能与miR-30a结合。在小鼠肾小管上皮细胞中lnc-PFDN表达下调后，miR-30a表达水平相对升高，而在小鼠肾小管上皮细胞中lnc-PFDN表达上调后，miR-30a表达水平相对下降。结果提示lnc-PFDN可能作为竞争性内源RNA (ceRNA)与miR-30a结合。本研究内容提示DN时lnc-PFDN可能作为ceRNA竞争结合miR-30a促进小鼠肾小管上皮细胞发生炎症和纤维化，干预lnc-PFDN可能成为改善DN的治疗靶点。