Screening And Functional Research Of The Interation Between Helicobacter Pylori CagA And Cellular Proteins In Gastric Mucosa

Helicobacter pylori{Hp) infects more than half the population not only in our country but also in the world. This infection is associated with chronic active gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Hp has been classified as a Group I carcinogen. And cytotoxin associated antigen A (CagA), a major Hp virulence factors, has been thought to be a bacterial oncoprotein. It has been reported that CagA could interact with more than 20 proteins, and activate much signaling, involved in development, movement, rearrangement of cytoskeleton, release of inflammatory molecules and so on, suggesting that CagA is closely related to gastric cancer.Although many partners of CagA have been discovered, the relation between CagA and gastric carcinoma is still not explained clearly. There may be some other partners remain unknown. In this study, yeast two-hybrid system was employed to screen partners in gastric mucosa for CagA. After confirming of the interaction between CagA and the candidate protein, we investigated the influence of this interaction on CagA in pathogenesis.The first part of this study is to screen the cellular proteins in gastric mucosa, which could interact with CagA by yeast two-hybrid system. Firstly, cagA was inserted into the pGBKT7 vector to construct the bait plasmid of yeast two-hybrid system. Then, the expression of BD-CagA fusion in yeasts was confirmed. And the intracellular β-galactosidase activities of the transformants were detected qualitatively and quantitatively. It was verified that CagA was not able to transactivate GAL4 responsive element. Secondly, total RNA was extracted from human gastric mucosa. Then, ds cDNA was acquired by reverse transcription and purification. After the ds cDNA and the linearized plasmid pGADT7-Rec were co-transformed into the competent yeasts, the yeast two-hybrid cDNA library of human gastric mucosa was successfully constructed. Lastly, a two-hybrid library screening was performed using the constructed cDNA library to seek for the cellular protein, which could interact with CagA. Four candidates were screened as followed:PGA3, eEF1A1, YWHAE and β-Actin.Based on the functional analysis of the above four candidates, YWHAE was selected and researched in the second part of this study in order to investigate the influence of the interaction between CagA and YWHAE on CagA in pathogenesis. Firstly, the interaction between CagA and YWHAE in yeast cells, in vitro and in mammalian cells was further confirmed by yeast two-hybrid, GST pull-down and co-immunoprecipitation, respectively. Secondly, the influence of the interaction between CagA and YWHAE on CagA in pathogenesis was investigated. pCMVTNT-cagA and pCMVTNT-YWHAE or YWHAE siRNA were co-transfected into SGC7901 cells, NF-κB activation was detected by a dual luciferase reporter assay. It was found that CagA could activate NF-κB through the interaction between CagA and YWHAE. Then, pCMVTNT-cagA and pCMVTNT-YWHAE or YWHAE siRNA were co-transfected into SGC7901 cells, IL-8 relative concentration was detected by ELISA. It was found that CagA could induce IL-8 secretion through this interaction. It was suggested that the interaction between CagA and YWHAE may participate in Hp CagA associated pathogensis.