microRNA-101a在尿酸诱导的肾小管上皮细胞转分化中的作用

目的研究尿酸对肾小管上皮细胞间充质转分化的影响及microRNA-101a在其中的作用。方法体外培养大鼠肾小管上皮细胞（NRK-52E），分别以0、10、100、500μmol/L的尿酸诱导细胞，采用实时定量PCR检测各组α-平滑肌肌动蛋白（α- SMA）、I型胶原（ColⅠ）及E钙蛋白mRNA的表达水平；Wersten blot检测各组中α- SMA的表达，免疫荧光检测各组中胶原蛋白I（COL）表达水平，观察细胞是否发生转分化。再用实时定量PCR检测各组中miR-101a及COX-2mRNA的表达水平，Wersten blot检测各组中COX-2的表达。进一步实验选择100μmol/L尿酸诱导细胞，并转染miR-101a mimics，分为3组，miR-101a组：先转染miR-101a mimics，再以100μmol/L尿酸培养基培养；空白对照组：以100μmol/L尿酸培养基培养；阴性对照组：细胞转染随机合成的miRNA NC片段，再以100μmol/L尿酸培养基培养。检测各组中α- SMA、ColⅠ、E钙蛋白及COX-2mRNA的表达量。结果培养基中尿酸浓度越高，α- SMA、ColⅠ表达水平也越高，E钙蛋白越低，提示细胞发生转分化，同时miR-101a表达减少，COX-2mRNA表达增多。miR-101a组与空白对照组和阴性对照组比较，α- SMA、ColⅠ及COX-2的表达明显降低（P＜0.05），E钙蛋白明显升高（P＜0.05），空白对照组与阴性对照组间差异无统计学意义（P＞0.05）。结论尿酸诱导肾小管上皮细胞转分化可能呈浓度依赖性， miR-101a及COX-2可能参与尿酸诱导肾小管上皮细胞转分化的调节过程。

Roles of microRNA-101a in epithelial-mesenchymal transition of rat renal tubular epithelial cells induced by uric acid

Objective To investigate the role of microRNA- 101a （miR- 101a） in epithelial- mesenchymal transition （EMT） of rat renal tubular epithelial cel s induced by uric acid. Methods Cultured renal tubular epithelial NRK- 52E cel s were treated with medium containing various concentration of uric acid （0μmol/L, 10μmol/L, 100μmol/L, 500μmol/L）. The expres-sion ofα- smooth muscle actin（α- SMA） mRNA and protein was detected by real time PCR and Western Blot, expression of col-lagen I （ColⅠ） mRNA and protein by real time PCR and immunofluorescence, and expression of E- cadherin mRNA by real time PCR. The expression of miR- 101a, cycloxygenase- 2（COX- 2） was detected by real time PCR or Western Blot. Before treated with 100μmol/L uric acid, the cultured NRK- 52E cel s were transfected with miR- 101a （miR- 101a group）, transfected with randomly synthesized miRNA （negative control group） or not transfected （blank control group）. Real time PCR was preformed to examine efficiency of transfection. Then the expression ofα- SMA, ColⅠ, E- cadherin and COX- 2 was determined. Results With the in-creasing of uric acid concentration, the expression of α- SMA and ColⅠ was increased,and E- cadherin decreased, while the expression of miR- 101a decreased and COX- 2 increased. The expression of α- SMA, ColⅠ and COX- 2 in miR- 101a group was significantly decreased, E- cadherin increases, compared with blank control group and negative control group （P〈0.05）, while there was no significant difference between the latter two groups. Conclusion Uric acid induces EMT of rat real tubular ep-ithelial NRK- 52E cel s in a concentration- dependent manner, miR- 101a and inflammation may be involved in EMT of NRK- 52E cel s induced by uric acid.