The Role And Mechanism Of FRA-1 In Helicobacter Pylori-infected Gastric Epthelial Cells

Objective: The purpose of this study is to analyze the expression of FRA-1 in gastritis and gastric cancer,and to further investigate the effect of H.pylori infection on the expression of FRA-1 in gastric mucosal epithelial cells,to explore the role and mechanism of FRA-1 in cancer progression after H.pylori infection,which is expected to provide new basis and ideas for clinical molecular diagnosis and targeted treatment of H.pylori infection-related gastritis and gastric cancer.Methods: The m RNA levels of FRA-1 in gastric cancer tissues and adjacent tissues of patients was analyzed by Oncomine database,and we also investigated the relationship between overall survival rate and FRA-1 expression.The protein levels of FRA-1 in gastritis and gastric cancer gastric tissues were detected by immunohistochemistry(IHC)at the level of clinical specimens.The expression of FRA-1 in H.pylori infected gastric epithelial cells lines and H.pylori infected cell models was detected by Western blot and q RT-PCR at the cell level.Specific small interfering RNA was used to knock down FRA-1 in GES-1 and MGC-803,then verified their efficiency.q PCR,western blot and ELISA were used to analysis the effect of FRA – 1 on H.pylori induced inflammatory cytokines: IL-1β,IL-6,IL-8 and TNFα in NF-κB pathway,and using western blot to detect the expression of phosphorylated NF-κB p65,NF-κB p65 and proteins in NF-κB pathway.Immunofluorescence analysis was used to detect the NF-κB p65 nuclear transfer.The effect of FRA-1 on the proliferation of gastric epithelial cells after H.pylori infection was observed by plate clone formation test.We performed transwell migration experiment to detect whether FRA-1 could affect the migration of gastric epithelial cells infected with H.pylori.We detected the change of EMT、proliferation and migration indexes,further analyze related protein expression of Wnt/β-catenin and TGF-β signaling pathway by western blot;the TGF-β was analyzed by immunofluorescence assay and nucleoprotein isolation,preliminarily analyze its role in the development of H.pylori related epithelial cells and its mechanism.Results:1.The results of the Oncomine database showed that compared with adjacent tissues,m RNA level of FRA-1 in gastric cancer tissues was higher,and the overall survival rate of gastric cancer patients with high FRA-1 expression was lower than that of gastric cancer patients with low FRA-1 expression.IHC results found the high expression of FRA-1 in gastritis and gastric cancer tissues.2.FRA-1 expressed in GES-1 and other gastric cancer cells.The expression of FRA-1 in gastric epithelial cells with positive H.pylori infection was significantly higher than that in gastric epithelial cells with negative H.pylori infection,and the increased expression was dependent on the live H.pylori contact.Specific small interfering RNA can successfully knock down FRA-1.3.Compared to H.pylori infection group,expression of IL-1β,IL-6,IL-8,TNFα and proteins in NF-κB p65 pathway were lower si-FRA-1 group after infected with H.pylori,shows that the silence of FRA-1 can accordingly inhibit the expression of inflammatory factors in NF-κB pathway,further suppresses H.pylori-induced NF-κB pathway and the nuclear transfer of NF-κB p65,thereby inhibition the inflammatory response induced by H.pylori infection.4.Plate clone formation test and transwell migration experiment pointed out that knocked down of FRA-1 in GES-1 and MGC-803 cells could inhibit cell proliferation and metastasis compared with that in H.pylori infection.The expression of PCNA,c-Myc,Cyclin D1 inhibited cells growth.It also increased the expression of TIMP1 while decreased MMP1、 MMP2 and MMP9,which inhibited the migration of tumor cells.What is more,the expression of E-cadherin(the epithelial cell marker)was increased,and the expression of N-cadherin、Vimentin、Snail、Twist1(the mesenchymal cell markers)were decreased,and it also inhibited the process of EMT.We also found that Wnt/β-catenin and TGF-β signaling pathway are related to it,and the related protein levels also changed correspondingly.Further inhibited the nuclear transfer of β-catenin.Conclusion: FRA-1 expression is increased in H.pylori infected tissues and cells.Downregulation of FRA-1 may protect the body from injury in H.pylori infection,and suppress the activation of NF-κB and the expression of downstream inflammatory factors caused by H.pylori.Also,FRA-1 may induce the occurrence and development of cancer after H.pylori infection by regulating the Wnt/β-catenin and TGF-β signaling pathway,which is expected to be a potential target for clinical molecular diagnosis of gastric cancer,targeted FRA-1 therapy may provide new ideas for the treatment of H.pylori-infected gastric cancer.