Tnf-α-inducing Protein Of Helicobacter Pylori Upregulates Nanog And Oct4 Of Gastric Cancer Stem Cells Via Wnt/β-catenin Signaling Pathway

Objective: The TNF-α-inducing protein(Tipα)is a newbacterial oncoprotein of Helicobacter pylori(H.pylori)and plays an important role in the tumorigenesis and development of gastric cancer.The aim of this study is to enrich and isolate the gastric cancer stem cells(GCSCs)from human gastric cancer cell line SGC-7901,further to investigate the effect of Tipα on the expression of GCSCs protein marker and stemness gene,as well as the role of Wnt/β-catenin signaling pathways in the stemness gene expression.Our findings will lay a foundation for further elucidation of the carcinogenic mechanism of H.pylori.Methods:In this paper,the Tipα was induced on a large scale and purified by Ni-NTA affinity column and identified by SDS-PAGE gel electrophoresis.Human gastric cancer line SGC701 were cultured by the method of serum-free suspending cultivation to enrich and isolate the cancer stem cells(CSCs)with sphere-forming.properties,then the m RNA and protein level of CSCs surface marker CD44,and stemness-related gene Oct4,and Nanog were detected by qRT-PCR and Western-blot.The ability of spheroid formation was observed under a light microscope.Different concentrations of Tipα were used to stimulate the SGC7901 cancer stem cells for different times,then the m RNA and protein level of CD44 were detected by q RT-PCR and Western-blot respectively.Furthermore,after stimulation by Tipα,Western-blot was employed to analyze the β-catenin phosphorylation(Ser675 and Ser552)in SGC7901 CSCs,and immunofluorscence assay was performed to detect the nuclear translocation of β-catenin.Morever,upon stimulation with Tipα,q RT-PCR was used to evaluate the m RNA expression level of Oct4 and Nanog in CSCs.Last,SGC7901 CSCs were pretreated with a specific inhibitor of Wnt/β-catenin signaling pathway,XAV939,followed by treatment with Tipα.Then the q RT-PCR and Western-blot were subsequently conducted to confirm the m RNA and protein expression level of CD44,as well as the downstream target genes(Oct4 and Nanog)of the Wnt/β-catenin signaling pathway.Results:1.We successfully enriched the the cancer stem cells with sphere-forming feature from human gastric cancer cell line SGC-7901 by the method of serum-free suspending culture,and the expression levels of CSC specific surface marker(CD44)and CSC genes(Oct4、Nanog)were obviously higher than that of SGC-7901 cells.2.We found the up-regulation of CD44 in Tipα-stimulated SGC7901 stem cells,and it is positively correlated with the concentration and stimulating time of Tipα.3.The β-catenin(Ser675 and Ser552)phosphorylation was observed in Tipα-stimulated SGC7901 stem cells.4.Tipα treatment increased the translocation of β-catenin from the cytoplasm to the nucleus in SGC7901 stem cells.5.The m RNA expression levels of CSC genes(Oct4,Nanog)were up-regulated in Tipα-stimulated SGC7901 stem cells.The optimal stimulation concentration and time of Tipα were 150 g/m L and 12 h,respectively.6.After the pretreatment of inhibitor XAV939,the expression of CD44 decreased in Tipα-stimulated SGC7901 stem cells,and the β-catenin(Ser675 and Ser552)phosphorylation and the expression of downstream target genes(Oct4 and Nanog)of the Wnt/β-catenin signaling pathway were inhibited.Conclusion:Tipα can upregulate the expression of Nanog and Oct4 in gastric cancer stem cells via Wnt/β-catenin signaling pathway.