CIP2A Expression Level Is Regulated By H. Pylori Infection Or Environmental Stimuli High Salt And EGCG

Helicobacter pylori (H. pylori) has been defined as a class I carcinogenic factor by WHO and its persistent colonization in stomach leads to an increased risk for peptic ulcers and gastric adenocarcinoma. Some H. pylori strains contain a 35-40 kb cag pathogenicity island (PAI) encoded by 27-33 genes. One constituent of the cag pathogenicity island (PAI) is called cagA which encodes a 120-140kDa CagA protein. CagA protein that has tyrosine phosphorylation (EPIYA) motifs is injected into the gastric epithelial cells by type IV secretory system (TFSS). cagA-positive H. pylori strains have a closer association with the progress of both peptic ulcers and gastric cancer than the cagA-negative strains according to epidemiological research. Within gastric epithelial cells, tyrosine phosphorylation occurs on the C-terminus of CagA by the Src family of tyrosine kinases. CagA interacts with many signal molecules and elicits a series of cellular events. Some changes related to cell morphology, cell scattering, cell proliferation and intercellular tight junctions have also been identified. According to these observations, bacterial oncoprotein CagA participates in gastric tissue injury caused by cagA-positive H. pylori infection.Unlimited cellular proliferation is a hallmark of tumors. Tumorigenesis is related to the disordered expression of some key factors which participate in the regulation of cell cycle progression, differentiation, apoptosis and senescence. Recent studies have reported that CIP2A or Cancerous Inhibitor of PP2A serves as an important oncoprotein. Protein phosphatase 2A (PP2A) can facilitate the proteolytic degradation of oncoprotein MYC and prevent malignant cell growth. More importantly, overexpression of CIP2A was observed in gastric cancer and its function was related to tumor cell proliferation. Btk family tyrosine kinase BMX was also involved in the promotion of tumor cell proliferation and regulation of cellular responses. Therefore, to elucidate the relationship between H. pylori infection and CIP2A expression could provide further evidence for understanding the mechanism of H. pylori carcinogenesis and CIP2A functions in gastric tumorigenesis.Gastric carcinogenesis is related to H. pylori infection, genetic susceptibility of hosts and environmental stimuli. High salt in food and drink is a very important risk factor which is involved in high incidence of gastric cancer. The close relationship between high salt in diet and gastric carcinogenic incidence has been proved through epidemic research and animal model. However, the mechanism of high salt intake-induced-gastric cancer high risk still remains unclear. For this reason, to investigate the enhancement effects of high salt on H. pylori infection-induced-CIP2A upregulation could help us to understand the molecular mechanism of synergistic effects between H. pylori infection and high salt stimuli in gastric carcinogenesis induction. As we all known, EGCG belongs to the most abundant polyphenol in green tea. The role of EGCG in the regulation of tumor cell proliferation has been verified in animal studies, human relevance and case-control reports. Up to now, the molecular mechanism of EGCG-inhibited-tumor cells proliferation still remains unclear. Therefore, to investigate the inhibitory effects of EGCG on CIP2A could provide further evidence for the understanding of the molecular mechanism of EGCG-induced-tumor inhibition.Gastric cancer is one of the most common tumors in worldwide and has a high incidence in China. The prognosis of gastric cancer is very poor because of the lack of credible early diagnosis and therapy. Biological carcinogen (H. pylori) and chemical carcinogen (salt and nitrite) are the factors involved in gastric carcinogenic induction. In contrast, polyphenols (green tea) and vitamins are the factors involved in gastric carcinogenic prevention. Molecular diagnostics and prediction for tumor occurrence has become a hot area of cancer research in recent years. It is still unknown whether both gastric carcinogenic induced factors and gastric carcinogenic inhibitory factors could act on the same target molecule. Our research mainly focused on the effects of H. pylori infection, high salt stimulus or EGCG treatment on tumor proliferation related protein CIP2A expression level. Our aim is to discover that CIP2A could act as the same target molecule of gastric carcinogenic induced factors and gastric carcinogenic inhibitory factors. The following is the main research data and experiment results.1. The upregulation effects of H. pylori infection on CIP2A expressionH. pylori virulence factor CagA interacts with many signal molecules and elicits a series of cellular events. These events include the Ras/mitogen-activated protein (MAP) kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway activation,β-catenin activation, Src kinase activation, NF-κB pathway activation, and p38 MAP kinase pathway activation. H. pylori infection could affect the expression level of gastric carcinogenic factors, such as MYC,β-catenin and Cyclin D1. But up to now, the relationship between H. pylori infection and CIP2A expression has never been studied. In order to elucidate the relationship between these two factors, we designed H. pylori infection and CagA transfection in gastric cells and tried to investigate whether main virulence factor CagA of H. pylori could increase the expression of oncoprotein CIP2A in cell lines. Furthermore, we used signal inhibitors to examine which pathway was involved in the CagA-mediated regulation of CIP2A expression. Overall, it is the first time the mechanism by which H. pylori infection and bacterial oncoprotein CagA upregulated CIP2A expression in gastric cell lines has been elucidated. CagA protein activated the Src and MEK/ERK signal pathways resulting in the elevation of expression of CIP2A protein in AGS cells. This work has contributed to understanding the mechanism of gastric tumors caused by H. pylori infection in humans.2. H. pylori infection-induced-CIP2A upregulation is enhanced by high saltIt has been elucidated in chapter 1 that H. pylori infection and bacterial oncoprotein CagA could upregulate CIP2A expression in gastric cell lines. These results provide further evidence for the understanding of the molecular mechanism of H. pylori infection-induced-gastric carcinogenesis. As mentioned above, high salt intake is one of the risk factors involved in gastric carcinogenesis. Besides that, high salt stimuli and H. pylori infection could act as synergistic factors for gastric carcinogenesis. In order to elucidate the effects of high salt stimuli on H. pylori virulence factors, and observe the enhancement effects of high salt stimuli on H. pylori infection-induced-CIP2A upregulation, we designed some analysis, including the analysis of H. pylori virulence factors after treatment by different concentrations of salt in cultures and the analysis of CIP2A expression level in cells after infection by H. pylori strains which has been treated by different concentrations of salt in cultures. The results showed that high salt stimuli could increase the expression level of H. pylori virulence genes (cagA, vacA, tlpB, tip-alpha). More important is that H. pylori infection-induced-CIP2A upregulation is enhanced by high salt. The data about CIP2A analysis make further evidence for the understanding of synergistic effects of high salt stimuli and H. pylori infection in gastric carcinogenesis.3. EGCG inhibits tumor cell proliferation through its inhibitory effects on CIP2AIt has been reported that the molecular mechanism of EGCG-induced-tumor inhibition is related to many key proteins, such as beta-catenin, Myc, Cyclin Dl, matrix metalloproteinases (MMPs), P53, etc. As we all known, both BMX/Etk tyrosine kinases and newly identified oncoprotein CIP2A belong to the key factors which could regulate tumor cell proliferation. Up to now, the relationship between tumor inhibitor EGCG, tumor promoter BMX kinases, and oncoprotein CIP2A has never been discovered. Therefore, the studies about the effects of EGCG on BMX and CIP2A, or interaction between BMX and CIP2A could help us to understand the molecular mechanism of EGCG-induced-tumor cell proliferation inhibition. Based on this purpose, western blot analysis was used to verify the effects of EGCG on BMX and CIP2A; Eukaryotic expression plasmid and siRNA were employed to observe the interaction between BMX and CIP2A; Clonogenicity analysis was performed to investigate the role of CIP2A in the EGCG-induced-tumor cell proliferation inhibition. In view of our results, it is the first time that we proved the inhibitory effects of EGCG on CIP2A and BMX. And we also verified that over-expression of BMX could increase the expression of CIP2A. Moreover we investigated the key factors which could be involved in the molecular mechanism of EGCG-inhibited-tumor cells proliferation. The studies provide further evidence for the understanding of the molecular mechanism of EGCG-induced-tumor inhibition, and also provide strong basis for the guidance of EGCG usage in clinical therapy.