乙型肝炎病毒X蛋白诱导肝癌细胞上皮-间叶样表型转化的实验观察

目的 探讨乙型肝炎病毒X蛋白(HBx)能否诱导肝癌细胞发生上皮-间叶样表型转化.方法 构建含HBx基因的重组腺病毒载体并转染肝癌细胞SMMC-7721,观察转染后细胞的形态变化,并通过RT-PCR、蛋白质印迹、免疫细胞化学等方法检测其上皮标志物和间叶标志物的表达变化,通过transwell实验评价其侵袭转移能力.结果 SMMC-7721细胞被含HBx基因的重组腺病毒载体转染1周后,多角形上皮样细胞表型转化为梭形成纤维细胞样表型,其上皮标志物E-cadherin表达为(0.37±0.05)与(0.74±0.07),α-catenin mRNA表达为(0.63±0.06)与(0.78±0.07)表达较对照组显著下调(P<0.05).β-catenin表达无明显变化(0.73±0.07)与(0.78±0.07)(P>0.05);间叶标志物N-cadhefin表达(0.78±0.04)与(0.60±0.07),Vimentin表达为(0.64±0.06)与(0.32±0.05),Fibronectin表达为(0.49±0.05)与(0.24±0.03)等与比对照组比较显著上调(P<0.05),并且侵袭转移能力显著提高.结论 乙肝病毒X蛋白可以诱导肝癌细胞SMMC-7721发生上皮-间叶样表型转化,并增强其侵袭转移能力.

Experimental study of epithelial-mesenchymal transition induced by HBx protein in liver cancer cell

Objective To explore whether or not HBx protein can induce epithelial-mesenchymal transition (EMT) in liver cancer cell SMMC-7721. Methods A recombinant adenovirus vector containing HBx gene was constructed and introduced into SMMC-7721 cell. The morphological changes were observed. The expression changes of epithelial and mesenchymal markers were investigated by RT-PCR, Western blot and immunohistochemistry. Invasive and metastatic capacity of transfected SMMC-7721 cell was evaluated through transwell migration assay. Results The recombinant adenovirus vector containing HBx gene was constructed and transfected successfully into SMMC-7721 cell. At Day 7 post-transfection, the transfected SMMC-7721 cell underwent the morphological change from a classic epithelial morphology to a spindle-like shape. In addition, the HBx protein significantly increased the expressions of N-cadherin, vimentin and fibronectin, but decreased the expressions of E-cadherin and α-catenin in SMMC-7721 cell. The cellular invasion assay indicated that transfection of HBx drastically enhanced the invasive potential of SMMC-7721 celL Conclusion HBx protein may induce EMT and enhance the invasive potential of SMMC-7721 cell.