| Background and objective:Helicobacter pylori(H.pylori)is a gram-negative bacterial pathogen and is colonized in gastric epithelium.Epidemiological studies indicated that H.pylori is the most common etiological agent for cancers that are related to infection.Global H.pylori infection rate is from 50% to 70% in the global population,Even though most individuals with H.pylori infection do not exhibit any clinical symptoms,long-term infection potentially leads to inflammation of gastric epithelium and peptic ulcers,but only about 1% infections develop to gastric cancer.The different outcome after H.pylori infection is mainly H.pylori virulence factors,together with the host genetic factors and environmental factors.Different H.pylori contains different virulence factors,divided into cagA positive strains and cagA negative strains.In recent years,studies have shown that infection of cagA positive strains have stronger virulence and pathogenicity.Wild type strains infection can lead to gastric epithelial cell ealy apoptosis and promote the cell proliferation and at the same time change the cell cycle.H.pylori 7.13 is cagA positive Cancer-causing strains and used to study pathogenic carcinogenic mechanism commonly.The purpose of this study is to construct 7.13 cagA gene knockout strains(△cagA)and wild type strains stimulate gastric epithelial cells GES-1 and gastric cancer cell AGS in vitro,observe the influence of wild type and △cagA stains on the cell apoptosis and proliferation and the cycle so to explore its possible mechanism.Preliminarily study the role of virulence factor CagA in gastric epithelial cells change by RNA-Seq and provide theoretical basis for the pathogenesis of gastric cancer.Methods:I.To insure the cagA gene of △cagA stains strains knock out successfully.1.Recovery and cultivate wild type and △cagA stains;2.Extract whole genome DNA of wild type and △cagA stains;3.The design of cagA gene primers for PCR amplification of cagA gene;4.Agarose gel electrophoresis to observe cagA gene to stripe;II.To verify that wild type strains can translocate CagA virulence proteins into the host cell:1.Gastric epithelial cells GES-1 and AGS co-culture respectively with wild type and △cagA stains with MOI=100/1(multiplicity of infection)fo 6 h,12 h,24 h,48 h,and set control group(without H.pylori);2.Cracking cells and extracted proteins of cells;3.Western blot detection the gastric epithelial cells GES-1 and AGS cell CagA protein content;III.The influence of wild type strains and △cagA strains on GES-1 and AGS cells biological functions:1.High content analysis GES-1 and AGS cells proportion apoptotic cells and cell cycle and so on.2.Real-time cell analysis cell proliferation of GES-1 and AGS cells stimulated by wild type and △cagA strains;IV.Effects of CagA on gastric epithelial cells transcriptome;1.Extract RNA of GES-1 cells stimulated by wild type and △cagA strains,set control group;2.RNA-Seq sample preparation and testing(dior company);3.Through the analysis of the enrichment,screen the expressed significant different genes which closely related to cell malignant transition(differentiation,proliferation,apoptosis,inflammation,etc.)signaling pathways and regulatory molecules Results:I.The result of wild type and △cagA strains cultivation and authenticate1.H.pylori grows well in bacterial culture palette,has form needlepoint transparent;2.PCR amplification and agarose gel electrophoresis results shown that △cagA strains cagA fragment without stripe,segments of wild type strains cagA purpose have stripes.II.To detect the contents of CagA protein in cells1.GES-1 and AGS cells were co-cultured with wild type and △cagA strains,CagA protein content in the cell is detected by western-blot,the wild type group detect the CagA protein,but the △cagA group and control group did not.2.Repeat three times quantitatively,CagA protein enter into the GES-1cells most is 6h and 12 h,and into the AGS cells is 12 h,24 h.III.The result of the impact of host cell function1.The results of the cell apoptosis:The apoptosis cell and necrotic cells proportion of GES-1 and AGS cells stimulated by wild type strains were higher than stimulated by △cagA strains(P< 0.05),and the most obvious difference apoptosis cell proportion was observed in 24 h.2.The results of the cell cycle:Compare to AGS cells and GES-1 cells stimulated by △cagA strains the control group,we can find that GES-1 and AGS cells stimulated by wild type strains show DNA synthesis cell percentage increased(P<0.05),mototit phase cell percentage decreases,and the most obvious difference was observed in 24 h.3.The effect on cell growth curve:Compare to GES-1 stimulated by △cagA strains and the control cells,GES-1 stimulated by wild type strains grow quickly,GES-1 stimulated by △cagA strains grow significantly than control group in the early time,but on the contrary in the late time.IV.Preliminarily study the CagA protein on host cell influence mechanism by transcriptome study:1.RNA-Seq genetic test found that there are gene changed in GES-1 cells stimulated by wild type and △cagA strains and the control group;2.Screen the expressed significant different genes and which closely related to cell malignant transition(differentiation,proliferation,apoptosis,inflammation,etc.)signaling pathways and regulatory molecules;3.GO through the analysis found that gene expression in different group GES-1cella have different levels change.Compare to control group,the gene number which regulate morphology,cell proliferation and growth,cell cycle,cell movement have different levels change in GES-1 cells stimulated by wild type strains,and the difference had statistical significance;Compare to △cagA strains group,the gene number which regulate cell morphology,differentiation,development and stress response have different levels change in GES-1 cells stimulated by wild type strains(P< 0.05),and the gene number which regulate cell cycle,proliferation and death process no statistical difference.4.Pathway annotations found that compared to control group,involved in P53 and NF-κB and Fox O and TLR and amino acid biosynthesis,HIF signaling pathway and vitamin B6 metabolism,transcriptional regulation,the oxidative phosphorylation process of cancer related genes have different degree of difference in GES-1 cells stilulated by wild type strains,and the differences were statistically significant(P<0.05),but the cell cycle gene differences was no statistically difference;Involved in the same signaling pathway in GES-1 cells stilulated by △ cagA strains,the differences were statistically significant(P< 0.05).Compared to △cagA strains infection group,Involved in JAK-STAT and Wnt and Hippo signaling pathways and the oxidative phosphorylation process related genes have different degree of difference(P< 0.05)in GES-1 cells stilulated by △cagA strains,but the cell cycle gene differences was no statistically difference(P>0.05).Conclusion:1.H.pylori virulence factor CagA can promote gastric epithelial cells early apoptosis necrosis and proliferation,but inhibition cell cycle and cell proliferation in late period;2.H.pylori virulence factor CagA can induce the m RNA expression profile of gastric epithelial cell,It regulates cell cycle,growth and apoptosis function gene changes and that may be refer to the mechanism of P53,the NF-κB,Wnt signaling pathway and so on. |
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