Preliminary Study. CagA Turn Transgenic Mouse Model And Pathogenic Mechanism

Helicobacter pylori (H. pylori), a microaerophilic, piral-shaped, lophotrichous Gram-negative bacterium, colonizes the stomach of approximately half of the world's human population and persists for the entire lifetime of the host unless treated. It has been evident for almost 30 years that H. pylor is involved in the development of gastric adenocarcinoma; the WHO proclaimed that H. pylori is a definite class I carcinogen in humans in 1994. Among gastroduodenal diseases caused by chronic infection with H. pylori are chronic atrophic gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma, peptic ulcers and gastric adenocarcinoma.The cagA-encoded CagA protein,one of major virulence factors in the pathogenesis of H. pylori, is the first bacterial oncoprotein to be identified in relation to human cancer. CagA protein is injected into gastric epithelial cells via the bacterial type IV secretion system, where it undergoes tyrosine phosphorylation by host cell kinases at the EPIYA motifs. CagA protein interacts with up to 20 cellular proteins and perturbs numerous host signaling pathways in a phosphorylation-dependent and phosphorylation-independent way, thereby promoting transformation of human gastric epithelial cells.H. pylori CagA proteins are distinguished from their structural diversity in C-terminal regions and can be classified into two major isoforms: East Asian CagA and Western CagA. This polymorphism is mainly due to differential combination and alignment of the four individual EPIYA segments. Notably, prevalent H. pylori strains isolated in China, Korea and Japan, where the incidences of gastric cancer are among the highest in the world, harbour the East Asian CagA isoform, whereas those strains isolated in Western countries (Europe, North America and Australia), where gastric cancer is infrequent, harbour the Western CagA isoform. The differential geographic distribution of differential CagA isoforms is studied, which may provided us with fundamental insights into large geographical variations in the incidence and mortality of gastric cancer in different parts of the world. To understand how CagA isoforms affect cellular functions of gastric epithelial cell, Phalanx HOA5.1 microarrays were employed to study the difference in global gene expression in AGS cells expressing CagA 9810 or CagA Ca52, and transgenic mice bearing cagA 9810^HS or cagA Ca52^HS were generated in this work. The present research includes the following three parts: 1. Optimization, synthesis and analysis of the humanized cagA gene.We chose two H. pylori strains: H. pylori 98-10 and H. pylori Ca52, which carry ABD CagA (East Asian CagA) and ABC CagA (Western CagA) ,respectively. Those cagA genes are characterized by A/T-rich sequences, which could induce rapid gene silencing in mammalian cells. Also, those cagA genes contain multiple ATTTA sequences that act as mRNA degradation motifs in mammalian cells. To avoid these potential problems, we converted bacterial cagA codons to optimal codons more commonly used in human genes according to CUTG, and got two optimized ORFs encoding CagA 9810 and CagA Ca52, respectively, which significantly reduced the A/T contents and eliminated the ATTTA sequences and removed cryptic splice sites. We synthesized cagA 9810^HS gene and cagA Ca52^HS gene by overlapping PCR. The both synthesized gene were 3′-tagged with a sequence encoding the hemagglutinin (HA) peptide to generate the humanized cagA gene (cagA^HS-HA): cagA 9810^HS–HA gene and cagA Ca52^HS–HA gene. The both synthesized cagA DNA (cagA^HS-HA) were cloned into the eukaryotic expression vector pcDNA3.1(+). Those recombinant plasmids were transiently transfected into AGS cells. The transcription level and intact protein expression of cagA^HS-HA were assayed by RT-PCR and Western Blot, respectively. The results revealed that the both synthesized cagA gene (cagA^HS-HA) were transcribed efficiently and expressed intact proteins.2. Effects of CagA isoforms (CagA 9810 or CagA Ca52) on global gene expression in AGS.Those recombinant plasmids (pcDNA3.1-cagA 9810^HS–HA and pcDNA3.1- cagA Ca52^HS ) were transfected into AGS cells, and G418-resistant clones were screened. AGS/cagA 9810^HS–HA and AGS/cagA Ca52^HS–HA stable transfected cell lines were confirmed by PCR, RT-PCR and Western Blotting.Total RNA was extracted from transfected AGS cells at 36 h post transfection using TRIzol. The Phalanx HOA5.1 microarrays, containing 30 275 probes, were employed to study effects of CagA 9810 and CagA Ca52 on global gene expression in AGS cells. Analyses of differentially expressed genes induced by CagA 9810 and CagA Ca52 showed that expression level of many genes in the AGS cells was changed, and the effected genes and intensity varied in two detected CagA isoforms. (1)With a filter of p≤0.01 and FC≥2, 121 genes were found to be regulated by CagA 9810 including 61 genes up-regulated and 60 genes down-regulated. The top ten genes up-regulated by CagA 9810 were LOC100287188,FPR3,PPT2,XIST,SERPINB2,FSCN1,C6orf222,FAM109B,ITGA2 and HOXB7 with fold changes ranging between 3.3 and 36.7; and the top ten down-regulated ones were NOTCH3,PLP1,H19,LY6D,TCF4,AMOT,CERK,SOHLH2,MAGED1 and FBXO2 with fold changes ranging between -4.8 and -9.3. (2) With a filter of p≤0.01 and FC≥2, 388 genes were affected by CagA Ca52 including 148 genes up-regulated and 240 genes down-regulated. The top ten genes up-regulated by CagA Ca52 were SEMA5A,OLR1,TPPP,AKR1C3,C12orf59,PRSS35,ANXA1,SLC40A1,DKK1 and HPGD with fold changes ranging between 3.8 and 9.6; and the top ten down-regulated ones were DHRS2,UGT1A10,FPR3,TUBA1A,SYNPO,RSAD2,WNT6,FBXO2,CD40 and MAP1B with fold changes ranging between -10.5 and -98.8. (3) With a filter of p≤0.01 and FC≥2, 44 genes were found to be regulated by both CagA 9810 and CagA Ca52. The simultaneously up-regulated genes were ACTG2,CCDC80,GNE,GPR126 and SARM1; and the simultaneously down-regulated genes were AMOT,ANK1,C5orf13,C9orf169,FBXO2,GPRC5D,H19,IGFBP2,KCNN4,KLK6,LGALS3BP,LY6D,MAP1B,NCAM1,NOTCH3,PLA2G2A,PLP1,PSG1,RENBP,SULT2B1,TCF4,TMEM187,TRIB3,TUBA1A,UGT1A10 and WNT6; and the genes reversely regulated were F5,NKAIN4,CA2,BIRC7,FPR3,DMBT1,FPR3,CA2,CREB3L3,DKK1,SEMA3C,CFTR and SEMA5A.The following functions were found to be significantly affected by CagA 9810 protein: response to mechanical stimulus,response to wounding,response to nutrient,response to extracellular stimulus,defense response and tube development. The following ones were found to be considerably affected by CagA Ca52 protein: response to steroid hormone stimulus,regulation of inflammatory response,regulation of response to external stimulus,circulatory system process,positive regulation of cell communication and response to organic substance. Among functions significantly affected by both CagA 9810 and CagA Ca52 are biological adhesion,blood circulation,branching morphogenesis of a tube,cell adhesion,cell motion,positive regulation of cell differentiation,respiratory tube development,response to extracellular stimulus,response to hormone stimulus,response to mechanical stimulus and wound healing.In this work, our data has provided us with systematical insights into effects of CagA on global gene expression in AGS.3. Generation of transgenic mice expressing CagA isoforms (CagA 9810 or CagA Ca52).With the expectation of predominant expression of CagA in the stomach, cagA 9810^HS or cagA Ca52^HS were connected downstream of theβsubunit gene promoter of mouse H+/K+-ATPase, respectively. After pronuclear injection of the transgenic constructs into fertilized mouse eggs, two lines of the cagA Ca52 ^HS transgenic mice and six lines of the cagA 9810^HS transgenic mice were established and confirmed by PCR and RT-PCR, respectively. These cagA^HS transgenic mice were indiscriminate from the wild-type mice in behavior and weight.In cagA Ca52^HS transgenic mice, cagA Ca52^HS mRNA was checked out in various organs and tissues. There're high levels of expression in stomach, liver, lung, spleen, intestine and kidney. This indicated leaky transcription from the promoter. The cagA 9810^HS transgenic mice are too little to get abnormal observations. However, at 9 months of age, cagA Ca52^HS transgenic mice were killed and autopsies showed a inflammation of gastric mucosa and a broad hyperplasia of gastric epithelial cell. And histological examination of gastric mucosa from 14-months old cagA Ca52^HS transgenic mice revealed an overt inflammation, extreme hyperplasia, intestinal metaplasia-like metaplasia and heteronuclear. It has been believed that these histological changes are important precancerous lesions.