论文部分内容阅读
目的研究肿瘤坏死因子α(TNF-α)对肝癌细胞上皮间质转化(EMT)的影响并探讨其分子机制。方法用TNF-α处理人肝癌细胞株(Hep3B和SMMC-7721)24h,倒置显微镜下观察处理前后细胞形态的变化,采用荧光定量RT-PCR和蛋白质印迹分析方法检测细胞中上皮细胞标记物上皮性钙黏蛋白(E-cadherin)和β-连环素(β-catenin)、间质细胞标记物波形蛋白(Vimentin)和神经性钙黏蛋白(N-cadherin)以及转录调控因子Twist的表达变化,Transwell及划痕实验分析细胞的侵袭能力。随后采用荧光素酶报告基因系统和细胞免疫荧光评估TNF-α对核因子κB(NF-κB)的激活作用;蛋白质印迹分析检测IκBα及p-IκBα蛋白表达水平。TNF-α联合应用NF-κB抑制剂后,检测细胞EMT表型变化。结果 Hep3B和SMMC-7721细胞用TNF-α处理后,表现EMT的表型;细胞的划痕愈合率明显大于未处理组(P<0.05),Tr-answell实验显示细胞穿透膜的数量亦显著增多(P<0.05)。TNF-α能有效激活NF-κB报告基因,促进NF-κB p65核转位及IκBα的磷酸化。应用NF-κB抑制剂BAY11-7082能够逆转TNF-α诱导EMT的作用(P<0.05)。结论 TNF-α通过NF-κB依赖性途径诱导肝癌细胞发生EMT,进而促进肝癌的侵袭转移。
Objective To investigate the effect of tumor necrosis factor-α (TNF-α) on epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells and to explore its molecular mechanism. Methods Human hepatocellular carcinoma cell lines (Hep3B and SMMC-7721) were treated with TNF-α for 24 hours. The morphological changes of the cells were observed under inverted microscope before and after treatment. The epithelial cell marker epitheliality was detected by quantitative RT-PCR and Western blot analysis E-cadherin, β-catenin, Vimentin and N-cadherin, as well as transcriptional regulatory factor Twist, and Transwell Scratch experiment analysis of cell invasion. Subsequently, the activation of NF-κB by TNF-α was assessed by luciferase reporter gene system and cellular immunofluorescence. The protein expression of IκBα and p-IκBα was detected by Western blotting. TNF-α combined with NF-κB inhibitor, the detection of EMT phenotype changes. Results Hep3B and SMMC-7721 cells treated with TNF-α showed the phenotype of EMT. The wound healing rate of Hep3B and SMMC-7721 cells was significantly greater than that of untreated cells (P <0.05). The number of cell penetrating membranes was also significantly increased by Tr-answell assay Increased (P <0.05). TNF-α can activate NF-κB reporter gene, promote NF-κB p65 nuclear translocation and IκBα phosphorylation. Application of NF-κB inhibitor BAY11-7082 reversed the effect of TNF-α-induced EMT (P <0.05). Conclusion TNF-α induces EMT in hepatocellular carcinoma cells through NF-κB-dependent pathway, and then promotes the invasion and metastasis of hepatocellular carcinoma.