The Impact Of ERK/MAPK Pathway In The Cell Effect Induced By CagA-YWHAE Interaction

Objectives: This study will explore whether CagA activates the MAPK pathway through interaction with YWHAE and its effect on cell effects,further improving the rearch for mechanism of CagA in gastric cancer,and providing new ideas for the prevention and treatment of H.pylori-related gastric cancer.Methods: 1 Determination of cell efficiency of CagA recombinant adenovirus infection Ad-CagA and Ad-GFP infected AGS cells at different concentrations to determine the optimal MOI value.Ad-GFP and Ad-CagA respectively infected YWHAE low expression(AGS-siYWHAE,YWHAE low expression cell line preserved in our laboratory)and YWHAE overexpression AGS cells(AGS-myc-YWHAE,the pcDNA3.1-myc-YWHAE plasmid constructed in our laboratory was transfected into AGS cells),and the expression of CagA protein was detected by Western blot.2 Determination of MAPK pathway activation by CagA-YWHAE interaction MAPK signaling pathway mainly includes: ERK,p38,JNK pathway 2.1 Western blot analysis of the effect of CagA-YWHAE interaction on MAPK pathway activation Ad-GFP and Ad-CagA respectively infected YWHAE low expression and overexpression AGS cells,and the expression levels of pERK,ERK,pp38,p38,pJNK and JNK were detected by Western blot.2.2 Validation of CagA-YWHAE interaction activating ERK/MAPK pathway with U0126(ERK upstream inhibitor) Ad-GFP and Ad-CagA respectively infected YWHAE low expression and overexpression AGS cells,and then treated with U0126.The expression levels of pERK and ERK were detected by Western blot.3 CagA-YWHAE interaction activates the effect of ERK/MAPK pathway on cellular effects 3.1 CagA-YWHAE interaction inhibits apoptosis depending on the ERK/MAPK pathway H.pylori strains NCTC11637 and NCTC11637?cagA infected YWHAE low expression cells,and apoptotic cells were detected by flow cytometry.U0126 pretreated YWHAE overexpression cells,and NCTC11637 and NCTC11637?cagA infected the above cells,and apoptotic cells were detected by flow cytometry.3.2 CagA-YWHAE interaction induces IL-8 expression in cells depending on ERK/MAPK signaling pathway The research team has initially verified the expression of IL-8 in cells induced by CagA-YWHAE interaction;U0126 pretreated YWHAE overexpression cells,and NCTC11637 and NCTC11637?cagA infected the above cells,and the expression of IL-8 was detected by ELISA.Results: 1 Determine the efficiency of CagA adenovirus-infected cells The Ad-CagA recombinant adenovirus amplified to the fourth generation was successfully obtained.The infection efficiency was the best when MOI=50.The results of Western blot showed that the CagA protein was expressed in both YWHAE low expression cells and YWHAE overexpression cells.2 Determine CagA-YWHAE interaction to activate ERK/MAPK pathway The phosphorylation level of ERK in Ad-CagA-infected AGS-siNC cells was significantly higher than that in Ad-GFP-infected cells(P<0.05).The phosphorylation level of ERK in Ad-CagA-infected AGS-siYWHAE cells was no significant difference compared with that in Ad-GFP-infected cells.ERK phosphorylation level in AGSsiYWHAE cells infected with Ad-CagA was significantly lower than that in AGS-siNC cells infected with Ad-CagA(P<0.05).Similar results were obtained with Ad-CagA and Ad-GFP infecting YWHAE overexpression cells.It is suggested that CagA-YWHAE interaction activates the ERK/MAPK pathway.Ad-CagA-infected AGS-siNC cells showed a significant decrease in ERK phosphorylation level in the U0126-treated group compared with the DMSO-treated group(P<0.05).Ad-CagA-infected AGS-siYWHAE cells showed no significant difference in ERK phosphorylation level in the U0126-treated group compared with the DMSO-treated group.Similar results were obtained with Ad-CagA and Ad-GFP infecting YWHAE overexpression cells.It was demonstrated that CagA-YWHAE interaction activates the ERK/MAPK pathway.Ad-CagA-infected AGS-siNC cells and AGS-siYWHAE cells both showed significantly higher p38 phosphorylation level than Ad-GFP-infected cells(P<0.05).However,Ad-CagA-infected AGS-siYWHAE cells had no significant change in p38 phosphorylation level compared with Ad-CagA-infected AGS-siNC cells.Similar results were obtained with Ad-CagA and Ad-GFP infecting YWHAE overexpression cells.It was shown that CagA can activate the p38/MAPK pathway but is not mediated by YWHAE.Ad-CagA-infected AGS-siNC cells and AGS-siYWHAE cells both had significantly higher JNK phosphorylation level than Ad-GFP-infected cells(P<0.05).However,Ad-CagA-infected AGS-siYWHAE cells had no significant change in JNK phosphorylation level compared with Ad-CagA-infected AGS-siNC cells.Similar results were obtained with Ad-CagA and Ad-GFP infecting YWHAE overexpression cells.It was shown that CagA can activate the JNK/MAPK pathway but is not mediated by YWHAE.3 CagA-YWHAE interaction activates the effect of ERK/MAPK pathway on cellular effects 3.1 CagA-YWHAE interaction inhibits apoptosis depending on ERK/MAPK pathway The apoptosis rate of NCTC11637-infected AGS-siNC cells was significantly lower than that of NCTC11637?cagA-infected cells(P<0.05).The apoptosis rate of NCTC11637-infected AGS-siYWHAE cells was no significant difference compared with that of NCTC11637?cagA-infected cells.The apoptosis rate of NCTC11637-infected AGS-siNC cells was significantly lower than that of NCTC11637-infected AGS-siYWHAE cells(P<0.05).It was shown that CagA-YWHAE interaction inhibit apoptosis.The apoptosis rate of AGS-myc-YWHAE cells and AGS-myc cells infected with NCTC11637 in the U0126-treated group was significantly higher than that in the DMSO-treated group(P<0.05).It was confirmed that the upstream inhibitor of ERK can effectively inhibit the anti-apoptosis effect of CagA-YWHAE interaction.3.2 CagA-YWHAE interaction induces IL-8 expression in cells depending on ERK/MAPK pathway The IL-8 expression of AGS-myc-YWHAE cells and AGS-myc cells infected with NCTC11637 in the U0126-treated group was significantly lower than that in the DMSO-treated group(P<0.05).It was confirmed that the upstream inhibitor of ERK can effectively inhibit the induction of IL-8 by CagA-YWHAE interaction.In conclusions:1.CagA-YWHAE interaction can activate ERK/MAPK pathway.2.CagA-YWHAE interaction inhibits apoptosis depending on the ERK/MAPK pathway.3.CagA-YWHAE interaction induces IL-8 expression in cells depending on the ERK/MAPK pathway.