Mechanisms For Helicobacter Pylori Cytotoxin Associated Gene A Protein Regulated α-Enolase In Gastric Carcinogenesis

Helicobacter pylori is a spiral-shaped, flagellated, micro-aerophilic Gram-negative bacillus first described in1982by Marshall and Warren. H. pylori is thought to colonize the gastric mucosa of more than50%of the world’s population, with the higher prevalence in the developing countries. Infection with Helicobacter pylori is the strongest risk factor for the development of chronic gastritis, gastric ulcer and gastric carcinoma, which is the second most common cause of cancer-related death worldwide. Some H. pylori strains possess a cytotoxin-associated gene (cag) pathogenicity island (cagPAI) that is present in about half of the Western strains and most of the Eastern strains. One constituent of the cag PAI is cagA, which encodes a120-140kDa CagA protein which is the most important bacterial oncoprotein.In order to illuminate the pathogenesis of H. pylori-induced gastric diseases, our group has developed a cagA gene knock out isogenic mutant strain. In our previous study, an in vitro model was established to characterize proteins differentially expressed in cagA positive H. pylori-infected gastric epithelial cells versus the cagA mutant strain. We found that a-Enolase (ENO1) expression level was increased in cagA positive H. pylori-infected cells as compared to mutant strain by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. ENO1is an isoenzyme of enolase, a key protein in the glycolytic pathway, which belongs to a novel class of surface proteins. Some evidences indicate a relation between ENO1and the progression of tumors, overexpression of ENO1is considered to take part in unlimited cellular proliferation of cancer, such as neuroendocrine tumors, neuroblastoma, lung cancer, and hepatocellular carcinoma. So ENO1has also been described as a new tumor-associated protein.However, up to now, the relationship between H. pylori infection and ENO1expression has never been studied. In the present study, we designed H. pylori infection and CagA eukaryotie egression plasmid pWT-cagA transfection in gastric epithelial cells and tried to investigate whether main virulence factor CagA of H. pylori could increase the expression of ENO1in vitro. Furthermore, we had constructed the CagA phosphorylation-resistant mutants to investigate whether phosphorylated-CagA is required for ENO1overexpression. Finally, we used signal inhibitors to examine which pathways wers involved in the CagA-mediated regulation of ENO1expression.Objective:Experimental researchs were undertaken to evaluate the effect of CagA on ENO1and its regulatory mechanism of ENO1expression upregulation.Materials and methods1. Extract of HP27genome, the full length cagA DNA (QD306710) was amplified by high-fidelity PCR, and then cloned into pMD19-T vector;2. cagA gene was digested by EcoR I and Xho I, cloned into pcDNA3.1(+) vector giving rise to pWT-cagA. Positive clones were selected for PCR, digestion and sequencing;3. We generated the CagA site-directed mutagenesis in which the tyrosine residues present in the B or D copies of the EPIYA sequences were replaced by cysteine by using splice overlap extension PCR. Specific primers were designed for B or D copies respectively. The phosphorylation-resistant plasmids were named PRCagA (B-) and PRCagA (D-) respectively;4. The WT-HP27(CagA+) strains were used to infect gastric cancer cells AGS and SGC-7901. The quantitative RT-PCR was designed to detect ENO1mRNA levels in which GAPDH gene was used as the reference gene. Western blot was used to detect ENO1protein expression.5. The CagA eukaryotic expression plasmid pWT-cagA and bland vector pcDNA3.1(+) were transfected into AGS cells. The bland vector transfected cell is control. After36h the total RNA of AGS cells were extracted by using Trizol reagent, EN01mRNA levels expression values were obtained by Realtime PCR. Western blot was used to detect ENO1protein expression.6. The phosphorylation-resistant expression plasmids PRCagA (B-) and PRCagA (D-) were transfected into AGS cells respectively. The blank vector transfected cells as control. ENO1mRNA levels expression were detected by Realtime PCR. Western blot was used to detect ENO1protein expression.7. To investigate associated signal pathways involved in CagA-mediated ENO1upregulation in the epithelial cells, we used the following reagents:BAY11-7082(5μM) to block NF-κB pathway, LY294002(50μM) to inhibit PI3kinase activity, PP1(10μM) to inhibit Src family kinase pathway, U0126(10μM) to inhibit both ERK1and ERK2kinase. One of these reagents to pre-incubate cells for1hand thentransfected pWT-cagA and pcDNA3.1(+)plasmids into AGS cells, respectively. ENO1mRNA levels expression values were obtained by Realtime PCR. Western blot was used to detect ENO1protein expression.Results1. PCR results show that:the cagA gene consists3510base pairs; the plasmid pMD19-T-cagA was constructed successfully;2. The CagA eukaryotic expression plasmid pWT-cagA were analyzed by EcoR I and Xho I double digestion. By PCR amplification from recombinant vector,3510bp of cagA was produced and further verified by sequencing;3. Mutation analysis was performed by direct sequencing. The DNA sequencing results revealed that the codon TAC which codes the tyrosine residues present in EPIYA sequences were replaced by cysteine (TGC). The CagA phosphorylation-resistant expression vector was constructed successfully;4. The Realtime PCR and Western blot assay showed that ENO1expression at mRNA and protein levels were dramatically increased in CagA positive H. pylori-infected AGS cells. Additionally, similar results were also observed in SGC-7901gastric cancer cells infection with WT-HP27(CagA+).5. The results showed that ENO1expression at mRNA level was increased in WT-CagA transfected cells compared to non-induced control cells and blank vector transformed cells. Western blot analysis showed that CagA could induce ENO1protein expression upregulation;6. The PR-cagvA plasmid encodes a CagA protein with a mutation in the EPIYA B motif or D motif, required for CagA tyrosine phosphorylation. After36h transfection of PR-cagA plasmid into AGS cells, ENO1mRNA and protein levels were unchanged in PR-cagA overexpressing cells as compared to control cells, the results indicated that ENO1upexpression required the CagA’s EPIYA motif tyrosine-phosphorylation;7. The western blot results showed that ENO1upregulation by CagA was attenuated by U0126and PP1; ENO1upregulation was maintained using BAY11-7082and LY294002. In total, the results indicated that MEK/ERK and Scr-family tyrosine kinase pathways participated in the up-regulation of ENO1by CagA.Conclusions1. H. pylori infection can induce ENO1mRNA and protein overexpression in gastric cancer cells;2. Upregulation of ENO1expression by H. pylori infection mediated by virulent factor CagA;3. The upregulation of ENO1protein level expression required the CagA’s EPIYA motif tyrosine-phosphorylation;4. H. pylori CagA activated the Src family kinase and MEK/ERK signal pathways, leading to ENO1upregulation.