The Interaction Between Helicobacter Pylori CagA And EEF1A1 In Human Gastric Cancer Cell As Well As Its Effect And Mechanism On IL-6 Expression

Helicobacter pylori（H.pylori）is closely related to the development of gastric cancer.Cytotoxin-associated gene A protein（Cag A）is the most important virulence protein of H.pylori,a kind of bacterial protein in cancer.Previous research suggested that Cag A may interact with eukaryotic transcription factor 1A1（e EF1A1）protein in Yeast two hybrid.Protein translation extension is one of classical functions of e EF1A1,also,e EF1A1 has many non-classical functions such as regulating cell cycle、proliferation and apoptosis,but the study of e EF1A1 in gastric cancer cells has not yet been carried out.Recently,German scientist found e EF1A1 can form complex with STAT3 and PKCδin gp130/JAK/STAT3 pathway,which can promote STAT3^S727phosphorylation and accelerate it entry into nucleus,and enhance Human Oncostatin-M（OSM）mediated IL-6 expression.IL-6 and its receptor has a close relevance with the incidence of gastric cancer development.H.pylori has been reported can induce the expression of IL-6.We speculated that H.pylori may induce expression of IL-6 with Cag A-e EF1A1.Research in this article consists of the following two parts:In the first part,we constructed pc DNA3.1-e EF1A1 plasmid with myc-tag,applied it to induce e EF1A1 overexpression in human gastric cancer cell AGS.Result of Co-immunoprecipitation（Co-IP）showed that Cag A not only interacts with over-expression e EF1A1,but also interacts with constitutive e EF1A1.We further construct truncated recombinant plasmid pc DNA3.1-e EF1A1-I,pc DNA3.1-e EF1A1-II,pc DNA3.1-e EF1A1-I+II,pc DNA3.1-e EF1A1-II+III,and Cag A truncated recombinant plasmid pc DNA3.1-Cag A-N,pc DNA3.1-Cag A-M,pc DNA3.1-Cag A-C,pc DNA3.1-Cag A-M+C,Co-IP showed that Cag A interacts with e EF1A1-Ithrough its M segment.The second part:Lentivirus was used to construct an AGS cell line with silenced e EF1A1 expression.q RT-PCR and Western Blotting（WB）experiments showed that the expression of e EF1A1 in the silenced AGS cells was reduced,and the e EF1A1silencing strain AGS-she EF1A1 and the silencing control strain AGS-C were successfully obtained（Negative control virus was transfected）.AGS-she EF1A1 and AGS-C were infected with H.pylori standard strain NCTC11637 and NCTC11637（35）cag A（cag A knockout strain）.The results showed that NCTC11637induced AGS-C expression of IL-6（18.68±1.23ng/ml）was significantly higher than NCTC11637（35）Cag A（12.02±1.52ng/ml）（P<0.001）.NCTC11637 induced AGS-C expressionofIL-6（18.68±1.23ng/ml）significantlyhigherthan AGS-she EF1A1（2.85±0.69ng/m L）（P<0.01）;NCTC11637 induced AGS-she EF1A1expression of IL-6（2.85±0.69ng/ml）and NCTC11637（35）Cag A induced AGS-she EF1A1no significant difference（3.72±0.93 ng/ml）（P>0.05）.These results indicated that Cag A and e EF1A1 co-mediate the expression of H.pylori infection induced IL-6.The Cag A fused virus particle Ad-cag A was constructed and confirmed by WB experiment.Ad-cag A infected AGS can express Cag A.The expression of IL-6 in AGS-C cells infected with Ad-cag A（28.55±2.20 ng/ml）was significantly higher than that of Ad-cag A infected AGS-she EF1A1 cells（17.39±1.63 ng/ml）（P<0.01）treated with OSM（100ng/ml,24h）.It further demonstrated that the interaction between Cag A and e EF1A1 enhanced IL-6 expression.AGS-she EF1A1 and AGS-C were infected with NCTC11637 and NCTC11637（35）cag A,and the supernatant culture medium was collected to detect IL-6.The infected cells were collected,and the cytoplasm and nuclear protein were extracted after lysing the cells.WB and image J software showed that the amount of p-STAT3^S727 in AGS-C nucleus was significantly higher than that in NCTC11637（35）cag A infection（P<0.01）.In AGS-she EF1A1 cells,NCTC11637 and NCTC11637（35）cag A has no significant difference in the amount of nuclear protein p-STAT3^S727 after NCTC11637（35）cag A infection.It was proved that Cag A and e EF1A1 together mediate the changes of p-STAT3^S727 in the nucleus.In addition,immunoprecipitation of cytoplasmproteins using the e EF1A1 antibody showed that the amount of e EF1A1binding to PKCδwas significantly higher in the AGS-C cytosol than in the NCTC11637（35）cag A infection（P<0.05）.The above experimental results show that after H.pylori infection,Cag A can promote the expression of IL-6 by promoting the recruitment of PKCδby e EF1A1 and increasing the amount of p-STAT3^S727 in the nucleus.Lentivirus was used to construct an AGS cell line with silenced STAT3 expression.It was found that the expression of AGS-sh STAT3 in NCTC11637 infection was significantly different from that in NCTC11637（35）cag A.WB experiment showed that the change of nuclear p-STAT3^S727 was closely related to IL-6,suggesting that p-STAT3^S727 mediated the expression of IL-6 in AGS cells.Conclusion:After H.pylori infection,Cag A can bind to e EF1A1-Ithrough M segment,thereby promoting the recruitment of PKCδin e EF1A1,and attenuating the nuclear inhibition of p-STAT3^S727 by PKCδin cytoplasm;and accelerating nuclear entry of p-STAT3^S727;the concentration of p-STAT3^S727 in the nucleus increased,which in turn promoted the expression of IL-6 in AGS cells.