| Gastric cancer is the second leading cause of cancer deaths worldwide. The prognosis for gastric cancer patients remains poor, especially in more advanced stages. Due to the lack of effective measures of early diagnosis and treatments, most patients are hard to found at an early age.So,it is essential to explore the mechanism of gastric carcinogenesis to seek for effective diagnosis marker and to improve therapeutic effect.Present study shows that chemokines and their receptors play important roles in many physiological and pathological processes, such as lymphocytes homing, immune responses, infection, autoimmune diseases, immunosuppression of the immunologic rejection and tumor cells proliferation, angiogenesis, migration and invasion.CXCL16belongs to CXC chemokines, CXCR6is its only receptor.This chemokine axis is high expressed in multiple tumors and promote or inhibit the progression of tumour. Gastric carcinogenesis is closely correlated with chronic inflammation. H.pylori can lead to chronic gastritis and promote the occurrence of peptic ulcer and gastric cancer. H.pylori is the world recognized class I carcinogen. Gastritis induced by H.pylori mainly causes the activation of Thl cells.This kind of cells secrete large amounts of IFN-y, which stimulates the secretion of various cytokines(including chemokines), eventually protect host through anti-bacterial and anti-tumor effect..Because chemokine involved in the physiological and pathological processes related to inflammation and tumor, this paper studied the impact of CXCL16 CXCR6axis on biological behavior of gastric cancer cell lines. At the same time, We contacted the correlation between H.pylori, IFN-y and CXCL16-CXCR6axis for H.pylori plays an important role in promoting chronic gastritis transform into stomach cancer. For a deeper analysis, we disscussed the possible mechanisms of CXCL16-CXCR6axis acting on gastric cancer, in order to provide theoretical support and guidance for the diagnosis and treatment of gastric cancer.Objective:To detect the expression of the human CXC chemokine ligand16(CXCL16) and its receptor CXCR6in the gastric carcinoma cell lines and gastric cancer tissues&adjacent non-neoplastic tissues clinically obtained, and to analyze the correlation between their expression and clinical pathological features. By using in-vitro assays to detect the influence of CXCL16-CXCR6axis on the migration, proliferation and clony forming ability of gastric carcinoma cells. And meanwhile test the expression changes of CXCL16and CXCR6after the stimulation of epithelial cell lines and gastric cancer cell lines by H.pylori and pro-inflammatory cytokine IFN-y. Build animal models of gastritis induced by H.pylori to detect the expression change of IFN-y and CXCL16and futher explore the correlation among H.pylori, IFN-γ and CXCL16and the mechanism of CXCL16-CXCR6axis acting on gastric cancer.Methods:1. Detected the expression of CXCL16and CXCR6in5kinds of gastric carcinoma cell lines and1kind of gastric immortalized cell lines.2. Embed the pathologic specimens(including gastric cancer tissues and adjacent non-neoplastic tissues) obtained at the tissue level in Paraffin to make slides, which were then put to detection for the expression of CXCL16&CXCR6in the tissue using immunohistochemistry methods. The expression of both chemokine ligand and its receptor in cancer tissues and adjacent non-neoplastic tissues were compared and analyzed the correlation between their expression and clinical pathological features.3. Small interfering RNA targeted at CXCR6was constructed. The highly aggressive MGC-803and moderately malignant BCG-823cell lines were selected for the transfections of the above-mentioned RNA. In vitro study, transwell migration assay, clone formation, CCK-8cell proliferation assay were adopted to observe the changes of migration, proliferation and clony forming ability of gastric cancer cells after the CXCL16/CXCR6axis was blocked.4. Treated the GES-1and MGC-803cell lines with H.pylori (with infect the cells with a rate of100:1bacteria to cells) and proinflammatory cytokine IFN-γ. The expression changes of the mRNA level and protein level of both CXCL16and CXCR6were tested respectively.5. Used the C57black mice to build animal models of gastritis induced by H.pylori and then detected the expression change of IFN-γ and CXCL16.Results:1. On mRNA level, the human CXC chemokine ligand16(CXCL16) and its receptor CXCR6were all expressed in the gastric carcinoma cell lines and the epithelial cell line as well, with no significant difference in their expression levels observed.2. The immunohistochemistry tests manifested that expression of CXCL16and CXCR6were detected both in gastric carcinoma glands and the corresponding adjacent normal tissues, primarily in carcinoma tissues and the gland cytoplasms and cell membranes in non-carcinoma tissues, and were also found in tumor infiltrated lymphocytes. The analysis demonstrated that the expression of the CXCL16/CXCR6in carcinoma tissues were higher than that in corresponding adjacent non-neoplastic tissues, with a meaningful statistical difference(CXCL16:p<0.01; CXCR6:p<0.01). Further analysis based on relevant clinicopathologic data demonstrated that the expression of CXCL16and CXCR6were cor-relationally related to the gastric gland carcinoma tumor size, differentiated degree, but not to the sex, age, lymph node metastasis and UICC stage of patients.3. Selected gastric cancer MGC-803and BGC-823cell lines as objects.The transwell migration assay, clone formation, CCK-8cell proliferation assay conducted after the CXCL16/CXCR6axis was blocked by transfection of siCXCR6revealed that the migration and clone formation capabilities and the proliferation ability of gastric carcinoma cell lines were evidently enhanced, indicating a restraining effect of CXCL16/CXCR6axis on the progression of gastric carcinoma.4. Selected Gastric epithelial cell line GES-1and gastric cancer cell line MGC-803as experimental objects, applied infectious stimulus from H.pylori and pro-inflammatory cytokine IFN-γ of different concentrations. The result demonstrated that there had been remarkable enhancement of the expression of both the mRNA level and the protein level of CXCL16. The enhancement of the expression of CXCL16with IFN-y treatment was correlational with the enhancement of the IFN-y concentration, while the expressions of CXCR6did not show any noticeable changes.5. Animal experiments showed that the expression of IFN-y and CXCL16in tissues infected by H.pylori raised obviously, and the increasing extent of their expression in the same mice were similar.Conclusion:CXCL16and CXCR6were expressed in gastric carcinoma glands, normal gastric glands and TILs. They were more frequently expressed in gastric cancer patients with a smaller tumor sizes and with highly and moderately differentiated adenocarcinoma. CXCL16-CXCR6axis had a probable restraining effect to the progression of gastric carcinoma. Both H.pylori and pro-inflammatory cytokine IFN-y could stimulate and enhance the expression of CXCL16. In vivo studies, the expression of IFN-y might be correlated with CXCL16. According to our experimental results, we concluded that in the microenviroment of gastritis and gastric cancer, there might exist a positive feedback loop in which IFN-y and CXCL16promote the expression of each other, the feedback loop could enhance inflammatory reaction and antitumor immune effect, which might be one of the mechanisms of CXCL16-CXCR6axis inhibiting the development of gastric cancer. |
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