miR-21在TGF-β1引起的肝卵圆细胞上皮间质转化中的作用

摘要

目的 探讨microRNA-21(miR-21)在TGF-β1引起的肝卵圆细胞(hepatic oval cell,HOC)上皮间质转化(epithelial mesenchymal transition,EMT)中的作用.方法 体外培养大鼠肝卵圆细胞,用10 ng/mL TGF-β1刺激HOC 1、3、5、7 d后,Western blotting检测EMT相关蛋白(E-cadherin、N-cadherin和Vi-mentin)表达情况;用10 ng/mL TGF-β1刺激HOC 5 d后,实时荧光定量PCR检测miR-21的表达情况;分别用miR-21增强和抑制慢病毒转染HOC构建稳定的细胞株,PCR检测miR-21的表达情况;接着用TGF-β1刺激miR-21抑制慢病毒转染的HOC 5 d,Western blotting检测E-cadherin、N-cadherin和Vimentin的表达;用miR-21增强慢病毒转染HOC后,Western blotting检测E-cadherin、N-cadherin和Vimentin的表达.结果 TGF-β1刺激HOC 1、3、5、7 d后,E-cadherin逐渐减低,N-cadherin和Vimentin逐渐升高.TGF-β1刺激HOC 5 d后miR-21的表达增强了4.32倍.分别用增强和抑制慢病毒转染HOC后,miR-21的表达增强了3.82倍和抑制了0.22倍.下调miR-21表达后,TGF-β1致HOC的EMT作用减弱.miR-21过表达后,HOC发生了EMT.结论 抑制miR-21的表达可以减少TGF-β1引起的HOC的EMT作用,从而减轻肝纤维化.%Objective To investigate the role of microRNA-21 (miR-21) in the epithelial mesenchymal transition (EMT) of hepatic oval cells (HOC) induced by TGF-β1. Methods Hepatic oval cells were cultured in vitro. The EMT-related proteins including E-cadherin, N-cadherin and Vimentin were detected by Western blotting after stimulation with 10 ng/mL TGF-β1 for 1, 3, 5 and 7 d. The expression of miR-21 was detected by real-time quantitative PCR with 10 ng/mL TGF-β1 for 5 d. The stable cell lines were constructed with enhanced and inhibited lentivirus transfection respectively and the expression of miR-21 was detected by PCR. The expression of E-cadherin, N-cadherin and Vimentin was detected by Western blotting after TGF-β1 stimulation on hepatic oval cells with miR-21 inhibited lentivirus transfection for 5 d. The expression of E-cadherin, N-cadherin and Vimentin was detected by Western blotting on hepatic oval cells with miR-21 enhanced lentivirus transfection. Results After the hepatic oval cells were stimulated by TGF-β1 for 1, 3, 5 and 7 d, the expression of E-cadherin decreased gradually, accompanying with gradually increased expression of N-cadherin and Vimentin. The expression of miR-21 in hepatic oval cells increased by 4.32 times after TGF-β1 stimulation for 5 d. The expression of miR-21 was enhanced by 3.82 times and decreased by 0.22 times respectively after the enhancement and inhibition of lentivirus transfection on hepatic oval cells. The down-regulated expression of miR-21 alleviated the EMT effect induced by TGF-β1on hepatic oval cells. After over-expression of miR-21, epithelial mesenchymal transition occurred in hepatic oval cells. Conclusion The inhibited expression of miR-21 can reduce the EMT effect induced by TGF-β1stimulation on HOC, thereby reducing liver fibrosis.