Yang Jie, Qin Guihui, Chen Junze
Department of Pharmacology, School of Pharmacy, Guangxi Medical University, Nanning 530021, China.E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2014 Dec;34(12):1743-7.
To explore the effect of lentivirus-mediated shRNA interference of Cx26 on epithelial-mesenchymal transition (EMT) and invasion of highly invasive human hepatocellular carcinoma cells in vitro.
SK-Hep-1 cells were infected with the lentivirus for delivering Cx26 shRNA, and the stably transfected cells were selected by puromycin. The interference efficiency of shRNA-Cx26 was assessed with real-time PCR and Western blotting. The morphological changes of the transfected SK-Hep-1 cells were observed microscopically, and the protein expressions of E-cadherin and vimentin were detected using Western blotting. The effect of Cx26 interference on the invasiveness of SK-Hep-1 cells was determined by Transwell invasion assay.
Compared with SK-Hep-1 cells infected with empty EGFP vector and uninfected cells, the cells transfected with shRNA-Cx26 showed significantly reduced mRNA and protein expressions of Cx26 (P<0.01), which resulted in obvious morphological conversion from mesenchymal cells to epithelial cells. shRNA-Cx26-transfected cells showed significantly increased E-cadherin protein expression (P<0.01) but decreased vimentin expression (P<0.01) with obviously attenuated invasive ability in vitro (P<0.01).
Targeted down-regulation of Cx26 expression can inhibit the EMT and invasion of SK-Hep-1 cells in vitro.
探讨慢病毒介导的Cx26基因短发夹RNA（shRNA）干扰对高侵袭性人肝癌细胞上皮-间质转化（EMT）及侵袭能力的影响。
用携带Cx26 shRNA的慢病毒感染SK-Hep-1细胞，并用嘌呤霉素筛选稳定转染的细胞。采用实时荧光定量PCR和蛋白质印迹法检测shRNA-Cx26的干扰效率。在显微镜下观察转染后SK-Hep-1细胞的形态变化，并用蛋白质印迹法检测E-钙黏蛋白和波形蛋白的表达。采用Transwell侵袭实验检测Cx26干扰对SK-Hep-1细胞侵袭能力的影响。
与感染空增强绿色荧光蛋白（EGFP）载体的SK-Hep-1细胞及未感染细胞相比，转染shRNA-Cx26的细胞Cx26的mRNA和蛋白表达显著降低（P<0.01），细胞形态由间充质样细胞明显转变为上皮样细胞。转染shRNA-Cx26的细胞E-钙黏蛋白表达显著增加（P<0.01），波形蛋白表达降低（P<0.01），体外侵袭能力明显减弱（P<0.01）。
靶向下调Cx26表达可抑制SK-Hep-1细胞体外EMT及侵袭能力。
