miR-17通过调控自噬影响TGF-β1诱导的肺纤维化

miR-17 Influences TGF-β1 Induced Pulmonary Fibrosis by Regulating Autophagy

目的探讨miR-17对转化生长因子-β1(TGF-β1)诱导的体外肺纤维化细胞模型自噬和纤维化的影响机制。方法重组人TGF-β1蛋白诱导人胚肺成纤维细胞HFL1,构建肺纤维化细胞模型,CCK-8检测细胞活力,ELISA试剂盒检测细胞上清中羟脯氨酸(Hyp)含量,鉴定造模成功与否。将细胞分成对照组、模型组、3-甲基腺嘌呤(3-MA)组、miR-17inhibitor组和miR-17inhibitor+3-MA组。CCK-8检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测线粒体自噬相关蛋白LC3-Ⅱ、p62和纤维化标志物α-SMA、CollagenⅠ蛋白的表达。结果TGF-β1诱导后HFL1细胞活力升高(P<0.01),细胞上清中Hyp含量升高(P<0.01),体外肺纤维化细胞模型构建成功。与对照组相比,模型组细胞活力显著升高(P<0.01),细胞凋亡率显著降低(P<0.01),细胞中LC3-Ⅱ蛋白表达水平显著降低(P<0.01),p62、α-SMA和CollagenⅠ蛋白表达水平显著升高(均P<0.01);与模型组相比,miR-17inhibitor组细胞活力显著降低(P<0.01),细胞凋亡率显著升高(P<0.01),细胞中LC3-Ⅱ蛋白表达水平显著升高(P<0.01),p62、α-SMA和CollagenⅠ蛋白表达水平显著降低(均P<0.01);与3-MA组相比,miR-17inhibitor+3-MA组细胞活力显著降低(P<0.01),细胞凋亡率显著升高(P<0.01),细胞中LC3-Ⅱ蛋白表达水平显著升高(P<0.01),p62、α-SMA和CollagenⅠ蛋白表达水平显著降低(均P<0.01)。结论抑制miR-17可通过激活自噬抑制TGF-β1诱导的肺纤维化。

Objective To investigate the effect of miR-17 on autophagy and fibrosis in transforming growth factor-β1(TGF-β1)induced pulmonary fibrosis cell models in vitro.Methods Human embryonic lung fibroblast HFL1 cells were induced with recombinant human TGF-β1 protein, and the pulmonary fibrosis cell model was established.CCK-8 was used to detect the cell activity, and the hydroxyproline(Hyp)content in the cell supernatant was detected by ELISA kit.The cells were divided into control group, model group, 3-methyladenine(3-MA)group, miR-17 inhibitor group and miR-17 inhibitor+3-MA group.Cell proliferation was detected by CCK-8,cell apoptosis was detected by flow cytometry, and the expressions of mitochondrial autophagy associated proteins LC3-Ⅱand p62,fibrosis markers α-SMA and collagenⅠprotein were detected by Western blotting.Results HFL1 cell activity increased after TGF-β1 induction(P<0.01),the content of Hyp in cell supernatant was increased(P<0.01),in vitro pulmonary fibrosis cell model was constructed successfully.Compared with the control group, the cell viability of model group was significantly increased(P<0.01),the apoptosis rate was significantly decreased(P<0.01),LC3-Ⅱprotein expression level was significantly decreased(P<0.01),the protein expression levels of p62,α-SMA and collagenⅠwere significantly increased(all P<0.01).Compared with the model group, cell viability of miR-17 inhibitor group was significantly decreased(P<0.01),the apoptosis rate was significantly increased(P<0.01),LC3-Ⅱprotein expression level was significantly increased(P<0.01),the protein expression levels of p62,α-SMA and collagenⅠwere significantly decreased(all P<0.01).Cell viability of miR-17 inhibitor+3-MA group was significantly decreased compared with 3-MA inhibitor group(P<0.01),the apoptosis rate was significantly increased(P<0.01),LC3-Ⅱprotein expression level was significantly increased(P<0.01),the protein expression levels of p62,α-SMA and collagenⅠwere significantly decreased(all P<0.01).Conclusion Inhibition of miR-17 can inhibit TGF-β1-induced pulmonary fibrosis through activation of autophagy.