Interaction Between CagA And YWHAE Activates NF-?B Pathway And Effect On Cell Invasion And Migration

Objective: Cytotoxin-associated antigen A(CagA)is an important virulence protein secreted by Helicobacter pylori(H.pylori)and plays an important role in the pathogenesis of gastric cancer.Previous studies showed that CagA can interact with many proteins in the host cells and activate multiple signaling pathways,suggesting that the interaction can affect cell proliferation,apoptosis,migration,invasion,and metastasis.We have shown previously that CagA can interact with the gastric mucosal cell protein YWHAE and activate NF-?B,however the specific activation pathway are not well understood.Thus,we aimed at investigating the specific pathway to activate NF-?B by interaction between CagA and YWHAE,and exploring the cell invasion and metastasis effects,in order to elucidate the pathogenic mechanism of CagA more completely.Methods 1 Construction of Ad-CagA adenovirusFirst,the recombinant plasmid pMD19-T-CagA was constructed,and then the recombinant shuttle plasmid pShuttle-IRES-hrGFP-CagA was constructed.Further,the principle of homologous recombination was used to construct the recombinant adenoviral plasmid pAd-CagA.pAd-CagA was transfected into 293 A cells and virus packaging was performed to obtain adenovirus Ad-CagA.AGS cells were infected with Ad-CagA and Ad-GFP in different concentration respectively,fluorescence microscopy was used to observe the expression of fluorescence and the optimal MOI value was determined,the expression of CagA protein was detected by Western blot.2 Determine the activation pathway of NF-?B in the interaction between CagA and YWHAEThe AGS-siNC cells were infected with Ad-GFP and Ad-CagA respectively,and stimulated with different concentrations of TNF-?.Western blot was used to detect the expression levels of p65 and p50 proteins in cytoplasm and nucleus in the NF-?B classical pathway to determine the optimal concentration of TNF-?.Quantitative detection of the activation of the classical pathway of NF-?B.The AGS-siNC and AGS-siYWHAE cells were infected with Ad-GFP and Ad-CagA respectively,and then treated with TNF-? in the optimal concentration,detecting the expression of p65 and p50 proteins in cytoplasmic and nuclear and detecting the level of p65 phosphorylation by Western blot;and the nuclear translocation of p65 was detected by immunofluorescence staining.3 Determine cell invasion and migration caused by the interaction between CagA and YWHAECells were seeded into the Transwell chamber after infected with adnovirus(spread Matrigel layer in the chamber two hours before the invasion was detected),and stained by crystal violet.The number of cells passing through the PET membrane was observed under microscope to detect the migration and invasion of cells;and the expression of epithelial-mesenchymal proteins,such as ?-catenin,Vimentin,Snail and Slug were detected by Western blot.4 Statistical analysis:Paired sample T test was used for data analysis by using SPSS22.0.Results 1 Construction of Ad-CagA adenovirus successfullyThe best infection efficiency was determined,Ad-CagA 50(MOI value),AdGFP 50(MOI value);the expression of CagA protein was detected by Western blot.2 The classical pathway of NF-?B activated by the interaction between CagA and YWHAEAfter the cells were stimulated with TNF-? in 0 ng/ml,1 ng/ml and 10 ng/ml,the induced p65 nuclear/cytoplasmic ratio was increased by 1.274±0.197,2.585±1.013,1.493±0.322 times respectively,the induced p50 nuclear/cytoplasmic ratio was increased by 1.793±0.403,1.950±0.115,1.093±0.524 times respectively in the Ad-CagA infected group compared with the Ad-GFP infected group,and there was a significant difference between the 1 ng/ml group(P<0.05).Thus,the concentration of 1 ng/ml was used for subsequent experiments.Quantitative detection of the activation of the classical pathway of NF-?B.The results showed that in AGS-siNC cells,the Ad-CagA-infected group induced a p65 nuclear/cytoplasmic ratio and a higher p50 nuclear/cytoplasmic ratio,and the level of p65 phosphorylation increased significantly,and significantly increasing the p65 nuclear translocation compared with the Ad-GFP-infected group(P<0.05).In AGS-siYWHAE cells,when YWHAE expression was inhibited,the Ad-CagAinfected group induced a p65 nuclear/cytoplasmic ratio and a p50 nuclear/cytoplasmic ratio increased,and the phosphorylation of p65 and the number of nuclear translocation of p65 increased compared to the Ad-GFP-infected group(P<0.05),however its ability to induce p65,p50 nuclear entry,the level of p65 phosphorylation,and p65 nuclear translocation was significantly lower than that of AGS-siNC cells(P<0.05),suggesting that CagA can interact with YWHAE to promote the activation of the NF-?B classical pathway.3 The effection of cell invasion and migration caused by the interaction between CagA and YWHAEThe results of Transwell showed that the number of cells migration and invasion in Ad-CagA infected group(286.3±67.03,528.3±130.4)was significantly increased compared with Ad-GFP infected group(168.3±52.34,242.3±48.81)in AGS-siNC cells(P<0.05).When YWHAE expression was inhibited,the number of cells migration and invasion in Ad-CagA infected group(171.87±59.68,355.07±103.54)was significantly increased compared with Ad-GFP infected group(126.8±65.04,187.13±39.5)in AGS-siYWHAE cells(P<0.05),but its ability to induce cells migration and invasion significantly decreased compared with AGS-siNC cells(P<0.05).The results show that CagA can promote cells migration by interacting with YWHAE.The results of Western blot showed that Ad-CagA infected group downregulated the expression of epithelial marker ?-catenin and upregulated the expression of mesenchymal markers Vimentin,Snail and Slug compared with AdGFP infected group in AGS-siNC cells(P<0.05);When YWHAE expression was inhibited,Ad-CagA infected group down-regulated epithelial marker ?-catenin and upregulated mesenchymal markers Vimentin,Snail and Slug compared with AdGFP infected group in AGS-siYWHAE cells(P<0.05),but its ability to upregulate the expression of mesenchymal markers Vimentin,Snail and Slug was significantly decreased compared with AGS-siNC cells(P<0.05),suggesting that CagA and YWHAE interaction can promote Epithelial to mesenchymal transition.Conclusion 1 Interaction of CagA and YWHAE activate the classical pathway of the NF-?B pathway.2 Interaction of CagA and YWHAE promote cell invasion and migration.3 Interaction of CagA and YWHAE promote Epithelial to mesenchymal transition.