TNF-α-Inducing Protein Of Helicobacter Pylori Induces Epithelial-mesenchymal Transition (EMT) In Gastric Cancer Cells Through Activation Of IL-6/STAT3 Signaling Pathway

[Objective] To evaluate the effects of Tipα on tumor process and reveal the underlying molecular mechanisms of EMT.[Methods] Firstly,the expression of recombinant Tipα protein was induced by IPTG,and then the protein was purified by Ni-NTA affinity column.The human gastric cancer cells line,SGC7901 cells,were cultured as conventional method.To explore the effects of Tipα on EMT progression,SGC7901 cells were treated with Tipα for 12 h,and the cells morphological changes were observed under a microscope.The protein and mRNA level of EMT-related molecules including E-cadherin,N-cadherin and Vimentin were detected by Western-blot and qRT-PCR.Immunofluorscence were used to analysis the cellular localization of Vimentin and STAT3 in SGC7901 cells.Furthermore,the level of IL-6 in cells was detected by ELISA.p-STAT3(Y705)and STAT3 protein expression changes induced by Tipα were analyzed by Western-blot.Wound-healing and Transwell assays were employed to determine migration potentials of SGC7901 cells.Cell proliferation was investigated by CCK8 assay.In addition,to explore whether Tipα promotes EMT through modulating IL-6/STAT3 pathways,the cells were pretreated by IL-6 antibody or STAT3 inhibitor SH-4-54 an hour prior to Tipα stimulation.Then the mRNA and protein expression of EMT-related molecules described above were detected by qRT-PCR and Western-blot.The change of cellular localization of Vimentin was analyzed by immunofluorscence.The invasion and migration of SGC7901 cells were evaluated by wound healing and transwell assay and the cell proliferation was measured by CCK8.[Results] We found 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 100μg/mL,which was therefore selected for the following experiments.Then we found that the expression level of E-cadherin reached minimal levels at 24 h and Vimentin reached maximal levels 12 h later.As a further confirmation,immunofluorescence staining also revealed that Vimentin expression on the cell membrane was in keeping with the above research results.The data of ELISA showed that treatment of SGC7901 cells with Tipα at the concentration of 100μg/ml for 0,1,3,6,9 and 12 h,the level of IL-6 was highly increased and peaked at 6h(P﹤0.01).In the doseand time-dependent experiment,the results of Western-blot indicated that STAT3(Y705)phosphorylation peaked at 100μg/mL post-treatment by Tipα for 3h.Furthermore,wound healing and cell migration assay results showed that the cell motilities on scratch wound or transwell migration significantly increased upon Tipα stimulation.The results of CCK8 assay also showed that,after the treament of Tipα,the proliferation of SGC7901 cells increased and peak at 48 h.When SGC7901 cells were pretreated with IL-6 neutralizing antibody or SH-4-54,the STAT3(Y705)phosphorylation was significantly inhibited(P﹤0.05).IL-6 antibody and SH-4-54 also reversed EMT markers expression in Tipα-stimulated SGC7901 cells(P﹤0.05).Compared with the control group,the cell proliferation and migration induced by Tipα were also alleviated by both agents(P﹤0.05).[Conclusion] Tipα induces EMT in gastric cancer cells via IL-6/STAT3 pathway activation.