【摘要】 目的研究IL-6/STAT3信号通路在幽门螺杆菌Tipα诱导胃癌细胞EMT形成中的作用。方法使用Tipα刺激胃癌SGC7901细胞,显微镜观察细胞形态变化;Western blot及qRT-PCR检测EMT标志物E-cadherin、N-cadherin和Vimentin的表达;免疫荧光检测STAT3的定位情况;ELISA检测IL-6水平;Western blot分析Tipα对SGC7901细胞p-STAT3和STAT3表达的影响。进一步应用IL-6中和抗体和STAT3抑制剂SH-4-54预处理细胞,以阻断IL-6/STAT3信号通路,继而检测通路阻断后对Tipα诱导的EMT形成的影响。结果 Tipα刺激SGC7901细胞12 h后细胞明显延长并出现伪足,EMT标志物N-cadherin和Vimentin表达增高,而E-cadherin表达减少。Tipα刺激能够增加IL-6分泌水平,且在6 h达峰值。Tipα可以浓度和时间依赖性方式诱导细胞p-STAT3水平增高。IL-6中和抗体及STAT3抑制剂预处理可逆转Tipα诱导的EMT形成。结论幽门螺杆菌Tipα可通过激活IL-6/STAT3信号通路诱导胃癌细胞EMT的形成。更多还原

【Abstract】 The aim of this study was to evaluate the role of IL-6/STAT3 signaling pathway in epithelialmesenchymal transition(EMT) of gastric cancer cells induced by TNF-α-inducing protein(Tipα) of Helicobacter pylori(H. pylori). Tipα protein was purified and used to stimulate the human gastric cancer cell line SGC7901 cells.Then the morphological changes of SGC7901 cell were observed under a microscope, and the protein and mRNA levels of EMT-related molecules including E-cadherin, N-cadherin and Vimentin were detected by Western blotting and qRT-PCR. Immunofluorscence was employed to analyze the localization of STAT3 in SGC7901 cells.Furthermore, the level of IL-6 was detected by ELISA,the expression levels of p-STAT3(Y705) and STAT3 protein were determined by Western blotting. In addition, SGC7901 cells were pretreated by IL-6 antibody or STAT3 inhibitor SH-4-54 an hour prior to Tipα stimulation, then we detected the expression changes of the EMT-related molecules and the change of cellular localization of STAT3. Data showed that Tipα-treated SGC7901 cells showed mesenchymal-like morphology changes and most of the cells elongated after treatment with Tipα for 12 h. The results of Western-blot and qRT-PCR showed that Tipα induced loss of epithelial marker E-cadherin, while up-regulation of mesenchymal markers N-cadherin and Vimentin at both protein and mRNA levels. The maximum response was observed at 100 μg/ml, which was therefore selected for following experiments. The expression level of E-cadherin reached minimal level at 24 h and Vimentin reached maximal level12 h later. The data of ELISA showed that IL-6 level was highly increased and peaked at 6 h after treatment of100 μg/ml Tipα. Western-blot indicated that STAT3(Y705) phosphorylation level peaked at 3 h post-treatment of100 μg/ml Tipα. Furthermore, when SGC7901 cells were pretreated with IL-6 neutralizing antibody or SH-4-54,the STAT3(Y705) phosphorylation was significantly inhibited. And both the agents also reversed EMT markers expression in Tipα-stimulated SGC7901 cells. Together, these results suggest that Tipα induces EMT in gastric cancer cells via IL-6/STAT3 pathway activation.更多还原