微小RNA-15b在腹膜间皮细胞转分化中的作用与机制

Role and mechanism of microRNA-15b in the regulation of epithelial-mesenchymal transition of peritoneal mesothelial cells

目的探讨微小RNA-15b（microRNA-15b，miR-15b）对人腹膜间皮细胞向上皮间充质转分化（EMT）的调节作用及可能的分子机制。方法使用138mmol／L高糖溶液刺激人腹膜间皮细胞株（HMrSV5）24h，观察高糖溶液对HMrSV5细胞miR-15b表达的影响；转染miR-15b模拟物（miR．15bmimic）使HMrSV5中miR-15b过表达或转染miR-15b抑制剂抑制HMrSV5中miR-15b的表达，再给予138mmol／L高糖溶液刺激24h，采用实时荧光定量PCR、Western印迹等方法，观察过表达miR-15b或抑制miR-15b表达对高糖诱导的HMrSV5细胞EMT相关蛋白和基因表达的影响。同时，观察调节miR-15b后，Smad7的mRNA和蛋白表达的改变。结果138mmol／L高糖溶液可使HMrSV5中miR-15b表达明显升高，转染miR-15b模拟物后可明显降低高糖诱导的上皮标志物E-cadherin的表达，并增加高糖引起的间充质标志物Vimentin及纤维化标志物Fibronectin mRNA和蛋白的高表达（均P〈0．05）；而转染miR．15b抑制剂则结果相反。同时，转染miR-15bmimic可明显抑制高糖诱导的Smad7mRNA及蛋白水平的上调（P〈0．05），而转染miR-15b抑制剂可明显增加高糖诱导的Smad7mRNA及蛋白水平的上调（P〈0．05）。结论miR-15b在高糖刺激腹膜间皮细胞HMrSV5转分化过程中上调，其可能通过调节TGF-β／Smad通路影响腹膜间皮细胞-上皮间充质转分化进程。miR-15b可能成为防治腹透相关性腹膜纤维化的新靶点。

Objective To explore the role and mechanism of microRNA-15b in the regulation of epithelial-mesenehymal transition （EMT） of human peritoneal mesothelial cells （HPMCs）. Methods PCR assay was used to determine the expression of microRNA- 15b in the HMrSV5 induced by 138 mmol/L high glucose for 24 h. MicroRNA-15b mimic or inhibitor was transfeeted into human peritoneal mesothelial ceils （HMrSVS） to over-express or down-regulate microRNA-15b. The cells were then incubated with 138 mmol/L high glucose for 24 h, and the expressions of E-cadherin（E-Cad）, Vimentin （VIM）, Fibronectin（FN） and Smad7 were detected by real-time PCR and Western blotting respeetively. Results microRNA- 15b in the HMrSV5 cells was over-expressed and down- regulated. Increased level of microRNA- 15b was obtained in HMrSV5 cells treated with high glucose. In vitro, high glucose led to the up- regulation of vimentin as well as fibronectin and the down-regulation of E- eadherin in HMrSV5 cells （all P 〈 0.05）, which indicated EMT and fibrosis. Suppression of microRNA- 15b by transfection with microRNA- 15b inhibitor partially reversed the EMT and fibrosis changes （P 〈 0.05）, while over - expression of microRNA - 15b by transfection with microRNA - 15b mimic obviously enhanced the EMT and fibrosis changes （P 〈 0.05）. Conclusions MicroRNA-15b mediates high glucose induced EMT in human peritoneal mesothelial cells by the inhibition of Smad7 possibly. MicroRNA- 15b maybe a new target for the prevention and treatment of peritoneal fibrosis during peritoneal dialysis （PD）.