miR-486在低氧诱导肺间质纤维化中的调控作用及机制

The effect and mechanism of MiR-486 in pulmonary fibrosis induced by hypoxia

目的探索低氧对人胚肺成纤维细胞(MRC5细胞)miR-486表达的调控作用,明确miR-486在低氧相关肺间质纤维化的发病中的调控作用及机制。方法将MRC5细胞进行低氧处理,利用qRT-PCR法检测miR-486、纤维连接蛋白(FN)及α-平滑肌肌动蛋白(α-SMA)的表达情况,用Western blot检测p-SMAD2及内参GAPDH蛋白的表达。用慢病毒技术将miR-486过表达或沉默后用qRT-PCR检测FN、α-SMA、胶原蛋白I型(CollagenⅠ)及CollagenⅢ的表达情况,用Western blot检测p-SMAD2及GAPDH蛋白的表达。结果低氧可抑制MRC5细胞中miR-486的表达,而促进FN、α-SMA及p-SMAD2的表达;过表达miR-486促进MRC5细胞miR-486的表达,而下调FN、α-SMA、CollagenⅠ、CollagenⅢ及p-SMAD2的表达;与之相反,沉默miR-486可以抑制MRC5细胞miR-486的表达,促进FN、α-SMA、CollagenⅠ、CollagenⅢ及p-SMAD2的表达。结论低氧可能通过调控miR-486影响p-SMAD2的表达,参与肺间质纤维化发病过程。

Objective To investigate the effect of hypoxia on miR-486 expression in human embryonic lung fibroblast MRC5 cells and identify mechanism of miR-486 in pulmonary fibrosis induced by hypoxia. Methods MRC5 cells were treated with hypoxia. Quantitative real-time PCR(qRT-PCR) was applied to detect the expression of miR-486,fibronectin(FN) and alpha smooth muscle actin(α-SMA),and Western blot was used to measure the expression of p-SMAD2 and GAPDH in MRC5. Lentivirus was used to mediate the overexpression or inhibition of miR-486 in MRC5 cells. Then,qQRT-PCR was used to detect the expression of miR-486,FN,α-SMA,Collagen Ⅰ and Collagen Ⅲ,and Western blot was used to measure the p-SMAD2 and GAPDH expression. Results Hypoxia inhibited the expression of miR-486 and promoted the FN,α-SMA and p-SMAD2 expression. MiR-486 overexpression successfully increased miR-486 expression and inhibited FN,α-SMA,Collagen Ⅰ,Collagen Ⅲ and p-SMAD2 expressions. MiR-486 knockdown upregulated the expressions of FN,α-SMA,Collagen Ⅰ,Collagen Ⅲ and p-SMAD2. Conclusion MiR-486 may regulate the pathogenesis of pulmonary fibrosis induced by hypoxia through targeting SMAD2.