摘要目的 探讨天龙咳喘灵提取物抑制转化生长因子β1(TGF-β1)诱导A549细胞上皮-间质转化(EMT)过程中微RNA(miRNA)的表达变化.方法 体外培养A549细胞,TGF-β1刺激A549细胞60 min后加入天龙咳喘灵提取物,应用相差显微镜观察A549细胞形态学变化.反转录-PCR、免疫印迹法检测α-平滑肌肌动蛋白(α-SMA) mRNA和蛋白表达.miRNA芯片检测A549细胞EMT前后miRNA表达的变化.结果 细胞形态学观察、反转录-PCR及免疫印迹检测均显示,5μg/L TGF-β1可以诱导A549细胞EMT且可以被50 mg/L天龙咳喘灵提取物抑制或逆转.miRNA芯片检测显示,与未处理的A549细胞比较,A549细胞EMT后有41种miRNA表达出现显著改变,其中26种表达上调2倍以上,15种表达下调2倍以上；与未处理的A549细胞比较,50 mg/L天龙咳喘灵提取物抑制TGF-β1诱导A549细胞EMT后有46种miRNA的表达出现显著变化,其中25种表达上调2倍以上,21种表达下调2倍以上；与TGF-β1诱导A549细胞EMT比较,50 mg/L天龙咳喘灵提取物抑制A549细胞EMT时有17种miRNA差异表达,其中13种表达下调2倍以上,4种表达上调2倍以上.结论 天龙咳喘灵提取物可以抑制TGF-β1诱导的A549细胞EMT,并参与调控miRNA的差异表达,可能有助于阻止慢性肺部纤维化疾病的进展.

abstractsObijective To investigate the alteration of miRNA expression in Tianlongkechuanling extracts-inhibited epithelial-mesenchymal transition (EMT) of A549 cells induced by transforming growth factor-β1 (TGF-β1).Methods A549 cells cultured in vitro were treated with TGF-β1 for 60 min following the addition of Tianlongkechuanling extracts.The phase-contrast microscope was employed to determine the changes in morphology of A549 cells.Reverse transcriptase polymerase chain reaction(RT-PCR) and Western blotting assay were adopted to detect α-smooth muscle actin(α-SMA) mRNA and protein expression.The miRNA chip was applied to assay the variation in miRNA prior to and following EMT of A549 cells.Results Findings of cell morphology,RT-PCR and Western blotting showed that EMT in A549 cells could be induced by 5 μg/L TGF-β1 and inhibited or reversed by 50 mg/L Tianlongkechuanling extracts.As suggested by miRNA chip test,(1) compared with untreated A549 cells,EMT resulted in significant altered expression of 41 miRNAs,including 26 miRNAs with a ≥2-fold up-regulation and 15 with a ≤2-fold downregulation; (2) compared with untreated A549 cells,the inhibitory effects of 50 mg/L Tianlongkechuanling extracts on TGF-β1-induced EMT resulted in significant altered expression of 46 miRNAs,including 25 miRNAs with a ≥2-fold up-regulation and 21 with a ≥2-fold down-regulation; (3) compared with TGF-β1-induced A549 cells,the inhibitory effects of 50 mg/L Tianlongkechuanling extracts on TGF-β1-induced EMT resulted in significant altered expression of 17 miRNAs,including 13 miRNAs with a ≥2-fold downregulation and 4 with a ≥2-fold up-regulation.Conclusion Tianlongkechuanling extracts effectively inhibit TGF-β1-induced EMT in A549 cells and are involved in differential expression of the miRNAs,and therefore,may help delaying the progression of chronic fibrotic pulmonary diseases.