| 摘要: |
| 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) 24 h,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),Transwell 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。 |
| 关键词: 肿瘤坏死因子α 肝细胞癌 上皮间质转化 肿瘤侵润 NF-κB |
| DOI：10.3724/SP.J.1008.2013.00271 |
| 投稿时间：2012-11-06修订日期：2013-02-05 |
| 基金项目:国家自然科学基金(81201584, 31171321). |
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| TNF-α induces epithelial-mesenchymal transition through NF-κB in hepatocellular carcinoma |
| KOU Xing-rui1,2,JING Ying-ying2,YU Guo-feng1,2,WU Meng-chao2,WEI Li-xin2* |
(1. Graduate Department of Soochow University, Suzhou 215006, Jiangsu, China
2. Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
*Corresponding author.) |
| Abstract: |
| Objective To investigate the role of tumor necrosis factor-α (TNF-α) in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells and the related mechanism. Methods HCC cell lines Hep3B and SMMC-7721 were treated with TNF-α for 24 h; then the cell morphological changes were observed by microscope and the expressions of the epithelial markers (E-cadherin and β-catenin), mesenchymal markers (Vimentin and N-cadherin), and EMT associated transcriptional factor Twist were examined by RT-PCR and Western blotting analysis. The invasion and metastasis ability was evaluated by Transwell and wound healing assay. Luciferase reporter assay and immunofluorescence were used to determine NF-κB transcriptional activity; Western blotting analysis was used to examine the expression levels of IκBα and p-IκBα protien. Cells was also incubated with TNF-α and NF-κB inhibitor (BAY11-7082) together, and then the phenotype of EMT was detected by microscope, RT-PCR and Western blotting analysis. Results Hep3B and SMMC-7721 cells had EMT phenotype after treated with TNF-α. Wound healing assay showed that the wound healing rate of cells exposed to TNF-α was significantly increased compared with the non-treated group (P<0.05), and Transwell assay showed that more cells penetrating the membrane after treatment with TNF-α (P<0.05). TNF-α effectively promoted IκBα phosphorylation and the subsequent NF-κB nuclear translocation. We also found that TNF-α-mediated EMT could be converted by NF-κB inhibitor (BAY11-7082) (P<0.05). Conclusion TNF-α induces EMT of HCC cells through NF-κB-dependent pathways, and subsequently promotes the invasion and metastasis of HCC. |
| Key words: tumor necrosis factor-α hepatocellular carcinoma epithelial-mesenchymal transition neoplasm invasiveness NF-κB |
