| Objective:The aim of this study is to evaluate the role of Wnt/?-catenin signaling pathway in Tip?-induced EMT in gastic cancer cells,and lay a foundation for further understanding the oncogenic mechanisms of Tip?.Methods:In this paper,the Tip? was induced on a large scale and purified by Ni-NTA affinity column.Next,we identify the Tip? by SDS-PAGE gel electrophoresis and Western-blot.Different concentrations of Tip? were used to stimulate the SGC7901 cells for different times,then the m RNA and protein level of epithelial molecules(E-cadherin?ZO-1)and mesenchymal molecules(N-cadherin?Vimentin)were detected by q RT-PCR and Western-blot.We stimulated SGC7901 cells with Tip? and used a microscope to observe the morphological changes of SGC7901 cells.Then,the ?-catenin phosphorylation(Ser675 and Ser552)in SGC7901 cells were analyzed by Western-blot.Later,immunofluorscence were used to detect the nuclear translocation of ?-catenin in SGC7901 cells.Next,SGC7901 cells were pretreated with XAV939,the inhibitor of the Wnt/?-catenin signaling pathway,then we treated the cells with Tip?.The Western-blot and q RT-PCR were subsequently used to detect the m RNA and protein expression level of EMT-related molecules and the downstream target genes(c-myc and cyclin D1)of the Wnt/?-catenin signaling pathway.Last,the ?-catenin phosphorylation was analyzed by Western-blot,and the migration of Tip?-stimulated SGC7901 cells were detected by would healing assay and transwell assay.Results:1.We found the down-regulation of E-cadherin and ZO-1 in Tip?-stimulated SGC7901 cells,while the expression levels of N-cadherin and Vimentin were up-regulated,and the possible optimal concentration of Tip? and the time point were 100 ?g/ml and 12 h respectively;2.The SGC7901 cells elongated after stimulated with the concentration of 100?g/ml of Tip? for 12h;3.The ?-catenin(Ser675 and Ser552)phosphorylation was observed in Tip?-stimulated SGC7901 cells in a time-dependent manner;4.Tip? led to the increased translocation of ?-catenin from the cytoplasm to the nucleus in SGC7901 cells;5.After the using of inhibitor XAV939,the expression of E-cadherin and ZO-1 in Tip?-stimulated SGC7901 cells increased,while the expression of N-cadherin and Vimentin,the ?-catenin phosphorylation and the downstream target genes(c-myc and cyclin D1)of the Wnt/?-catenin signaling pathway were reduced;6.Wound healing assay and transwell assay showed that Tip? increased the migration of SGC7901 cells,while the use of XAV939 resulted in impaired migration in both wound healing assay and transwell assay.Conclusion:1.Tip? can activate Wnt/?-catenin signaling pathway;2.Tip? induces EMT in gastric cancer cells via Wnt/?-catenin signaling pathway. |
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