| Gastric cancer is one of the major malignant tumors harmful to human’s health, and the incidence and mortality of gastric cancer remain high worldwide. According to the latest statistics, the incidence and mortality of gastric cancer rank second in all tumors in China. The number of deaths from gastric cancer is up to 50 million each year. Therefore, it is significant to study the factors of tumorigenesis and progression in gastric cancer.Currently, Helicobacter pylori(H.pylori) is the most common human pathogen worldwide, infecting an estimated 50 % of the global population, and in some areas up to 80 %(Management of Helicobacter pylori infection-the Maastricht IV/ Florence Consensus Report, 2012). In our country, H.pylori infection rate is also high, and the infection rate is up to 40 %- 60 % in adult. The World Health Organization declared H.pylori to be a Group I human carcinogen for gastric cancer in 1994. Although H.pylori infection rate is high in the population, only part of those infected patient eventually develop gastric cancer. It is because the pathogenicity of H.pylori is related to the kinds of H.pylori pathogenic strains, host genetic factors and environmental factors and so on. Different types of H.pylori strains is an important reason leading to different clinical outcomes. Maastricht consensus mentioned that the development of gastric cancer is influenced by H.pylori virulence factors and the level of evidence based medicine is 1a. Cytotoxin-associated gene A(CagA) as one of the important virulence factors, the positive strains infection lead to severe clinical consequences, and strain with CagA is an important pathogenic strains. CagA is encoded by cag pathogenicity island(cag PAI), which is a 40 kb DNA fragment. CagA, which is injected into host cells through type IV secretion system(T4SS), behaves as a bacterial oncoprotein. The latest evidence based medical results show CagA~+ H.pylori increases the risk of gastric cancer. CagA transgenic mice significantly increased the incidence of gastric cancer, confirming CagA is a key molecule to promote gastric carcinogenesis.Cancer stem cells(CSCs) theory believe that there is a population of cancer cells with stem cell-like properties, which possess the ability to unlimited self-renewal and the ability to induce tumorigenesis. CSCs are thought to be seed cells of tumorigenesis. Because CSCs also have high metastatic potential and resistance to chemotherapy, CSCs is considered a key factor related to the recurrence and metastasis of malignant tumor.Previous studies focused on H.pylori-promoted proliferation and H.pylori-inhibited apoptosis of gastric cancer. But the lastest study found that CagA~+ H.pylori can induce epithelial mesenchymal transition(EMT) of gastric cancer cells and generates gastric cancer cells with CSCs properties via EMT-like changes. This study further confirms the role of CagA~+ H.pylori-promoted cancer stem cell-like properties in gastric cancer cells and elucidates its mechanism. Therefore, this study enriches the pathogenesis of CagA~+ H.pylori strain infection and help to deep understand the role of CagA~+ H.pylori in gastric carcinogenesis and progression, which provide a new theoretical basis for specific eradication CagA~+ H.pylori.【Objectives】1. To build the model of H.pylori-infected gastric cancer cells, to evaluate the role of CagA~+ H.pylori-promoted cancer stem cell-like properties in gastric cancer cells.2. To explore the mechanism of CagA~+ H.pylori-promoted cancer stem cell-like properties in gastric cancer cells.【Methods】1. To evaluate the role of CagA~+ H.pylori-promoted cancer stem cell-like properties in gastric cancer cells.(1) To build the model of H.pylori-infected gastric cancer cells;(2) Flow cytometry analysis was used to test the proportion of gastric CSC surface markers positive gastric cancer cells in CagA~+ H.pylori–infected group(H.p ylori strains NCTC 11637), CagA- H.pylori–infected group(CagA knockout strains) and uninfected group.(3) PCR was used to test the mRNA level of gastric CSC surface markers in the above groups.(4) Spheroid formation assay was used to evaluate the ability of spheroid formation of gastric cancer cells in the above groups.(5) Western blotting(WB) was used to detect the expression level of essential factors for maintenance of self-renewal and pluripotency in the above groups.(6) Light microscope was used to observe the morphology change of gastric cancer cell in the above groups, and WB was used to detect the expression of EMT markers.2. To explore the mechanism of CagA~+ H.pylori-promoted cancer stem cell-like properties in gastric cancer cells.(1) Immunofluorescence was used to observe the localization of β-catenin in the above groups.(2) Dual luciferase reporter assays were used to detect the activation of Wnt/β-catenin signaling pathway in the above groups.(3) WB was used to detect the expression of β-catenin Ser675 and Ser552 at different time points in CagA~+ H.pylori-infected or CagA- H.pylori-infected gastric cancer cells.(4) Gastric cancer cells were pre-treated with a c-met inhibitor(SU11274) or a PI3K/Akt inhibitor(GSK) and then infected with CagA~+ H.pylori. WB was used to detect the expression of β-catenin Ser675 and Ser552.(5) Gastric cancer cells were pre-treated with Wnt/β-catenin inhibitor(XAV939) and then infected with CagA~+ H.pylori. Flow cytometry analysis was used to test the proportion of gastric CSC surface markers positive gastric cancer cells, PCR was used to test the mRNA level of gastric CSC surface markers, and spheroid formation assay was used to evaluate the ability of spheroid formation of gastric cancer cells.(6) Gastric cancer cells were pre-treated with Wnt/β-catenin inhibitor(XAV939) and then infected with CagA~+ H.pylori. WB and PCR were used to detect the expression of Nanog and Oct4.(7) We constructed two vectors containing either the promoter of Nanog or the promoter of Oct4. Dual luciferase reporter assays were used to detect the promotor activities of Nanog and Oct4 in the above groups.(8) The bioinformatics was used to predict potential binding sites for β-catenin in the promoters of Nanog and Oct4. Chromatin immunoprecipitation(ChIP) was used to detect the binding of β-catenin to Nanog and Oct4 promoters.(9) C13 urea breath test and serological anti-CagA analysis were used to investigate patients diagnosed with gastric cancer were infected with CagA~+ H.pylori or CagA- H.pylori. Immunohistochemistry and PCR were used to detect the expression of Nanog and Oct4 in gastric cancer tissues.【Results】1. CagA~+ H.pylori-infected gastric cancer cells exhibit cancer stem cell-like properties, compared with uninfected cells or CagA- H.pylori-infected cells.2. CagA~+ H.pylori activates Wnt/β-catenin signaling pathway, including nuclear accumulation and transcriptional activation of β-catenin.3. The activation of Wnt/β-catenin signaling pathway is dependent upon the CagA~+ H.pylori-mediated phosphorylation of β-catenin at the C-terminal Ser675 and Ser552 residues in a c-met- and/or Akt-dependent manner.4. Wnt/β-catenin signaling pathway activation is responsible for CagA~+ H.pylori-induced cancer stem cell-like properties5. Activation of Wnt/β-catenin signaling is involved in CagA~+ H.pylori-induced upregulation of Nanog and Oct4.6. CagA~+ H.pylori infection increases the expression of Nanog and Oct4 in gastric cancer tissues, compared with CagA- H.pylori infection.【Conclusions】CagA~+ H.pylori upregulates Nanog and Oct4 via Wnt/β-catenin signaling to promote cancer stem cell-like properties in gastric cancer.【Significance】At present, the guidelines recommend high-risk population(chronic atrophic gastritis, ulcers, intestinal metaplasia, dysplasia and postoperative gastric cancer patients etc.) to eradicate H.pylori, but its molecular mechanism is unclear. This study confirms the highly virulent pathogenic strains of H.pylori-- CagA~+ H.pylori can promote CSC-like properties, which may be an important reason for gastric epithelial cells from dysplasia to the eventual gastric cancer, may also be an important reason for recurrence and metastasis of gastric cancer. Therefore, this study provides a new perspective on the pathogenesis of H.pylori infection and help to deep understand the role of CagA~+ H.pylori in gastric carcinogenesis and progression, which provide a new theoretical basis for specific eradication CagA~+ H.pylori. |
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