微小RNA-612抑制低氧诱导的肾小管上皮细胞上皮-间充质转化作用及其分子机制

Inhibitory effect of microRNA-612 on hypoxia-induced epithelial mesenchymal transition of renal tubular epithelial cells and the molecular mechanism

目的探讨微小RNA（miR）-612对低氧诱导的肾小管上皮细胞转分化的影响及其分子机制。方法分别以100μmol／L的氯化钴（CoCl2·6H2O，Co）处理体外培养的HK2人肾小管上皮细胞株6h、12h和24h，检测低氧对细胞miR-612表达的影响。将HK2细胞分为对照组、单纯低氧组（100txmol／LCo处理24h）、低氧＋10nmol／LmiR-612转染组和低氧＋30nmol／LmiR、612转染组，检测各组miR-612的表达，采用Westernblot检测低氧诱导因子-1α（HIF-1α）、α-平滑肌激动蛋白（α-SMA）和上皮钙黏素（E—cad）的表达。预测、验证miR-612的靶基因，并检测miR-612表达水平变化对靶基因蛋白及其下游蛋白表达的影响。结果co造成的低氧处理导致细胞中miR-612表达显著降低（P〈0．05）；Co处理24h，miR-612表达水平下降约80％以上（P〈0．05）。10nmol／LmiR-612转染导致低氧诱导的α-SMA表达水平下降约50％，E-cad的表达水平显著上升，30nmol／LmiR-612转染产生的上述效果更明显。miR-612可靶定WNTl0AmRNA，并抑制WNT10A蛋白及β连环蛋白（β-catenin）的表达。结论MiR-612抑制低氧诱导的肾小管上皮细胞转分化，该作用可能通过抑制WNT10A介导的WNT／β-eatenin通路实现。

Objective To investigate the effect of microRNA （ miR）-612 on hypoxia-induced epithelial mesenchymal transition （EMT） of renal tubular epithelial cells and the potential molecular mechanism. Methods 100 μmol/L cobalt chloride （CoC12 6 H2O, Co） was used to incubate the cultured HK2 human renal tubular epithelial cell line for 6h, 12h and 24h. The impact of hypoxia on the expression of miR-612 was examined. HK2 cells were divided into 4 groups：the control group, the hypoxia group （ the cells in this group were treated with 100 μmol/L Co for 24h ）, the hypoxia ＋ 10 nmol/L miR-612 transfection group and the hypoxia ＋ 30 nmol/L miR-612 transfection group. The expression of miR-612 was detected. Western blotting was used to detect the protein levels of hypoxia inducible factor-1α （ HIF-1 α） and α-smooth muscle actin （α-SMA） and E-cadherin （ E-cad ）. The target gene of miR-612 was predicted and confirmed. The effect of miR-612 level change on the expression of the target gene protein and the downstream proteins was also detected. Results Co causing hypoxia resulted in a significant decrease in the expression of miR-612 （ P 〈 0. 05 ）. The expression level of miR-612 was decreased by more than 80% when treated with Co for 24 h（P 〈 0.05 ）. 10 nmol/L miR-612 transfection resulted in a decrease of about 50% of the expression level of hypoxic-induced α-SMA and an increase of the expression level of E-cad. The effect of 30 nmol/L miR-612 transfection was more obvious than that of 10 nmol/L miR-612. MiR-612 can target WNT10A mRNA and inhibit the expression of WNT10A protein and β-catenin. Conclusion MiR-612 inhibits hypoxia-induced EMT of renal tubular epithelial cells, which may be mediated by inhibition of WNT10A mediated WNT/β-catenin pathway.