慢病毒介导hsa-miR-199a-5p通过转化生长因子-β/Smad通路抑制瘢痕疙瘩成纤维细胞纤维化的研究

Inhibitory effect of hsa-miR-199a-5p mediated by lentivirus on keloid fibroblast fibrosis through TGF-β/Smad pathway

目的探讨hsa-miR-199a-5p对瘢痕疙瘩成纤维细胞(KFs)纤维化过程的影响及其与转化生长因子(TGF)-β/Smad信号通路的关系。方法将携带hsa-miR-199a-5p基因的慢病毒载体(hsa-miR-199a-5p组)及空白慢病毒载体(rLv-NC组)感染KFs, 并设空白对照组(KFs正常培养)。采用CCK-8法检测各组KFs的增殖情况。慢病毒感染KFs 48 h后, 应用实时定量PCR(qRT-PCR)检测各组KFs的hsa-miR-199a-5p表达水平, 流式细胞仪检测细胞凋亡情况, Transwell实验检测细胞侵袭能力, 并分别采用qRT-PCR和蛋白质印迹法检测Ⅰ、Ⅲ型胶原、α-平滑肌肌动蛋白(α-SMA)、Smad3及TGF-β1的mRNA和蛋白表达水平。结果病毒感染KFs 48 h后, hsa-miR-199a-5p组的hsa-miR-199a-5p表达量明显高于rLv-NC组和空白对照组, 差异有统计学意义(P<0.01);与rLv-NC组和空白对照组相比, hsa-miR-199a-5p组的细胞增殖率、侵袭性均有所降低, 细胞凋亡率有所升高, 差异有统计学意义(P<0.01);hsa-miR-199a-5p组的Ⅰ、Ⅲ型胶原、α-SMA、Smad3、TGF-β1的mRNA及蛋白表达水平均明显低于rLv-NC组和空白对照组, 差异有统计学意义(P<0.01)。结论 hsa-miR-199a-5p可能通过抑制TGF-β/Smad通路的表达, 抑制KFs增殖与侵袭, 并促进KFs凋亡, 从而抑制瘢痕疙瘩纤维化。

Objective To investigate the effect of hsa-miR-199a-5p on keloid fibroblasts(KFs)fibrosis and its relationship with TGF-β/Smad signaling pathway in vitro.Methods KFs were infected with the lentiviral vector carrying hsa-miR-199a-5p(hsa-miR-199a-5p group)or the blank vector(rLv-NC group),and a blank control group was set.The proliferation status of KFs were detected by CCK-8 assay.At 48 hours after infection,the expression of hsa-miR-199a-5p in KFs were detected by quantitative real-time PCR(qRT-PCR);cell apoptosis was detected by flow cytometry analysis;cell invasion ability was detected by Transwell;The mRNA and protein levels of collagenⅠ,collagenⅢ,α-smooth muscle actin(α-SMA),Smad3 and TGF-β1 were detected by qRT-PCR and Western blotting,respectively.Results At 48 hours after infection with RLV-hsa-miR-199a-5p lentiviral vector,the mRNA expression level of hsa-miR-199a-5p in hsa-miR-199a-5p group KFs was significantly higher than that in rLv-NC group and blank control group,and the difference was statistically significant(P<0.01).Compared with the rLv-NC and blank control groups,the proliferation rate and invasiveness of the hsa-miR-199a-5p group decreased,the apoptosis rate increased,with statistical significance(P<0.01);the mRNA and protein expression levels of collagenⅠ,collagenⅢ,α-SMA,Smad3 and TGF-β1 in hsa-miR-199a-5p group were significantly lower than those in rLv-NC group and blank control group,with statistical significance(P<0.01).Conclusions hsa-miR-199a-5p may inhibit the proliferation and invasion of KFs by inhibiting TGF-β/Smad pathway,and promote the apoptosis of KFs,thus inhibiting keloid fibrosis.