miR-200及其靶基因ZEB1/2在急性肺损伤后早期肺纤维化中上皮-间质转化(EMT)过程

The Effect of miR-200 and Their Targets ZEB1/2 on Epithelial-mesenchymal Transition(EMT) in Early Pulmonary Fibrosis Caused by Acute Lung Injury

目的观察脂多糖(lipopolysaccharide,LPS)诱发小鼠急性肺损伤后早期肺纤维化过程中miR-200b/c及其靶基因ZEB1/2的表达变化.方法应用LPS 3次打击的方法构建小鼠急性肺损伤后早期肺纤维化模型,分别于造模后第3、7、14、21天处死小鼠,留取肺组织备用.各组小鼠肺组织切片行HE和Masson染色并在光学显微镜下观察病理改变;Real-time PCR检测肺组织miR-200b、miR-200c、ZEB1 m RNA、ZEB2 m RNA表达;Western blot检测肺组织ZEB1、ZEB2、E-cadherin、Vimentin、α-SMA蛋白表达.结果 (1)病理结果:与对照组相比,LPS处理后第3天肺组织胶原纤维开始沉积,随着时间延长,肺纤维化程度逐渐加重;(2)Real time-PCR结果:随着肺纤维化程度加重,miR-200b、miR-200c水平均呈下降趋势,第7、14、21天时均显著低于对照组(P<0.01);ZEB1 m RNA、ZEB2 m RNA水平呈上升趋势,且ZEB2 m RNA较ZEB1 m RNA表达增加更显著;(3)Western blot结果:随着急性肺损伤后肺纤维化的进展,ZEB1、ZEB2蛋白表达亦升高,与其m RNA表达变化相一致;上皮标志物E-cadherin蛋白表达逐渐减少,间质标志物Vimentin、α-SMA蛋白表达逐渐增多.结论在LPS诱发急性肺损伤后早期肺纤维化过程中miR-200b/c表达降低,并通过负性调控其靶控基因转录抑制因子ZEB1/2的表达促进上皮向间质转化.

Objective To observe the expression profiles of miR-200b/c and their targets ZEB1/2 in early pulmonary fibrosis caused by acute lung injury induced by lipopolysaccharide in mice. Methods Early pulmonary fibrosis caused by acute lung injury is built via a lipopolysaccharide three-hit regimen. Mice were sacrificed at post-injective day 3, 7, 14, 21 respectively and the lung tissue specimens were collected. The lung tissue sections were stained with HE and Masson staining and pathological changes were observed by optical microscope. The expression profiles of miR-200b, miR-200e, ZEB1 mRNA and ZEB2 mRNA were detected by real-time PCR. Western blot was utilized to detect the levels of ZEB1, ZEB2, E-cadherin, Vimentin, ct-SMA proteins. Results （1） pathological findings： compared with the control group, the collagen fibers deposited on on the third day after LPS treatment and pulmonary fibrosis gradually worsened; （2） Real time-PCR results： With the aggravation of pulmonary fibrosis, miR-200b and miR-200c levels were declined and the levels of miR-200b/c at post-injective day 7, 14, 21 were significantly lower than that of control group （P〈0.01）. ZEB1 mRNA and ZEB2 mRNA levels were gradually increased in the process of pulmonary fibrosis and the increased magnitude of ZEB2 mRNA was more significant than that ofZEB1 mRNA; （3） Western blot results： ZEB1 and ZEB2 protein levels were also gradually increased, consistent with their mRNA levels and the expression of E-cadherin protein was decreased while Vimentin and α -SMA protein levels increased with the evolution of pulmonary fibrosis caused by acute lung injury. Conclusion miR-200b and miR-200c promote epithelial-mesenchymal transition by inhibiting their targets ZEB1/2 in early pulmonary fibrosis caused by acute lung injury induced by LPS.