The Phenotype Induced By The Expression Of CagA In Specific Tissue Of Drosophila And The Influence To The Expression Of Some Certain Members Of Wnt Signaling Pathway

PrefaceGastric carcinoma is the fourth most common cancer the scond cause of cancer-related deaths worldwid,with approximately 700,000 deaths worldwide each year.The epidemiological studies have indicated that Helicobacter pylori plays a key role in the developments of gastric carcinomas.H.pylori is a gram-negative, spiral-shaped bacteium.It is seperated and cultured by Warren and Marshall in 1984. Based on the presence or absence of cagA,H.pylori can be divided into cagA-positive and cagA-negative strains.H.pylori cagA positive strains inject CagA into the cytoplasm of the cells via the typeⅣsecretion system.Translocated CagA then localizes to the inner leaflet of the plasma membrane,where it undergoes tyrosine phosphorylation by Src family kinases(SFKs) or Abl kinase.The tyrosine phosphorylation site of CagA is characterized by the presence a Glu-Pro-Ile-Tyr-Ala (EPIYA) motif,which is present in multiple numbers in the carboxy-terminal polymorphic region(EPIYA-repeat region) of the protein.The research about cagA transgenic mice confirm that CagA is a bacterial oncoprotein which can induced the gastrointestinal carcinomas and hematopoietic malignancies and it is tyrosine phosphorylation-dependent.CagA also destabilizes the E-cadherin/β-catenin complex, a major component of the adherens junctions,and thereby deregulates theβ-catenin signal.β-catenin is a major component of the adherens junctions,and it is also the key effector of Wnt signaling pathway.The Wnt signaling pathway is evolutionary conserved and Wnt signaling is involved in normal development,as well as development of a variety of human malignancies.But the studies about the relation between Wnt signaling pathway and gastric adenocarcinoma are still insufficient. Whether CagA.as a tumorigenesis factor of gastric adenocarcinoma affects the Wnt signaling pathway need be studied.In this research,I used the GAL4-UAS system to check the eye phenotype and wing phenotype of the cagA expressing fly.And examine the effect of CagA to the expression of some important members(Wingless,Armadillo and APC) of Wnt signaling pathway in drosophila.Methods1.The construct of pUAST-cagA-HA plasmidThe polymerase chain reaction.was done in order to get the cagA-HA fragment from the pSP65SRα-cagA-HA(Add BamHI and XhoI enzyme site).Digeste with BamHI and XhoI and ligate it with pUSAT,which has been digested by BglⅡand XhoI. The pUAST-cagA-HA plasmid was amplified by DH5αand then the plasmids were digested with EcoRI or BamHI/XhoI under standard conditions and Agarose Gel Electrophoresis of extracted recombinants were done to confirm appropriate sizes of the DNAs.Sequencing was done to confirm whether the sequence is correct.2.The process of construct of cagA transgenic flyCollect the embryos of yw(yellow body and white eye) for 30 minute intervals just before injection and immerse in bleach(available chlorine 2.5%) for about 1 minute to dechorionate.Line up the embryos on agar plate straightly.Stick the embryos on the cover slide and put the slide in drying box for 5-10 minutes.Cover embryos with HC oil,and then inject the embryo from rear end with the plasmid.For the reason of technique of operating,we could not get the transgenic fly for 3 monthes.Professor Karen Guilemin(from Institute of Molecular Biology,University of Oregon,Eugene,Oregon,USA) got the cagA transgenic fly recently.After communication,Professor Karen Guilemin kindly sent the cagA transgenic fly (yw;UAS-HA-cagA),All the cagA transgenic fly used in the experiments in this paper are from this line.3.The preparation of the flies expressing CagA in the eyes or in the wingsCross 5-10 GMR-GAL4 male flies with 5-10 UAS-HA-cagA virgins and culture at 25℃.Check the eye phenotype of F1 flies.Pick about 10 pairs of F1 flies into a new tube to produce F2 generation.And check the phenotype of F2 flies.Cross the GMR-GAL4 male flies and yw virgins,F1 flies are as control.Cross 5-10 MS1096-GAL4 virgin flies with 5-10 UAS-HA-cagA male flies,and culture at 25℃, check the wing phenotype of F1 flies.Cross the MS1096-GAL4 virgin flies and yw male flies,F1 flies are as control.4.The preparation of the flies expressing CagA in the embryos and detecting the expression of wingless,APC and Armadillo by immunofluorescence techniqueCollect the F1 male flies from Arm-GAL4 crossing with UAS-HA-cagA,cross them with UAS-HA-cagA virgins.Collect the embryos produced within 10h.After dechorionating with bleach,fix the embryos with fixation solution for 30 minutes.After BBT washing,incubate the embryos in primary antibody(mouse anti-Wingless,rat anti-HA,rabbit ant-EAPC mouse anti-Armadillo) at 4℃overnight.Incubate the secondary antibody(Alexa goat 488 anti-rabbit,546 anti-mouse,647 anti-rat,1:1000) for 2 hours at RT.The slide was made with mounting medium and the images were taken by using a Nikon A1 with C1 EclipseTi confocal microscope.Results1.The construct of pUAST-cagA-HA is successfulThe electrophoresis results after the enzyme cutting showed the cagA-HA fragment had been inserted into the pUAST.The integrated squencing results were blasted in NCBI website.The result showed the sequence was identical with cagA gene of H pylori strain NCTC11637.2.The eye and wing phenotype induced by the CagA in drosophilaGMR-GAL4 male flies crossed with UAS-HA-cagA virgins.All the F1 flies showed slightly rough-eye phenotype and some of the F2 flies showed strongly rough-eye phenotype.MS1096-GAL4 virgins crossed with UAS-HA-cagA male flies.All the F1 flies showed curving-wing phenotype3.The expression of wingless,APC and Armadillo in the CagA-expressing embryos showed no differenceIn the CagA expressing embyos,the width of wingless strip did not show diffence compared with control group.The expressions of APC and Armadillo in the CagA-expressing embryos showed no difference.Conclusions1.CagA can affect the development of organs in drosophila and induced the phenotype change in eyes and wings though it is from bacterium.2.EPIYA A-B-C-C type CagA has no effect to the expression of Wingless,APC andβ-catenin in drosophila.