The Mechanism Research Of Protein Interaction Between14-3-3ε And RKIP On The Development Of Gastric Cancer

BackgroudGastric cancer is one of the common malignant tumors in our country, and its mortality ranks the second after the long cancer. At present, the incidence of the gastric cancer is the fourth of the tumors in the world, and the incidence and mortality rate of it is the first among the malignant tumor s of digestive tract. Gastric cancer is the current major diseases that endanger people’s health. The development of gastric cancer which is a complex pathological process involved in its pathogenesis is still not clear. No matter looking for new tumor markers and gene therapeutic targets, or clarifying the mechanisms of the development of gastric cancer, would play an important role in its early diagnosis and treatment.We have used18O stable isotope labeling and LCM in the quantitative proteome analysis of clinical gastric adenocarcinoma in our previous experiments. A total of78differential proteins were identified between the gastric adenocarcinoma and nonmalignant epithelial cells. We first found RKIP were over-expressed in normal gastric mucosa but down-regulated in gastric adenocarcinoma. The expression of RKIP protein is closely related to the development and the tumor differentiation of gastric cancer by clinical research. On the basis of this work, we intend to offer the application of targeted proteomic techniques, which take affinity purification coupling mass spectrometry techniques as the principal thing, to isolate and identify the RKIP protein interactions in SGC7901gastric cancer cells. And14-3-3εis one of interacting proteins.14-3-3protein family which possesses powerful protein binding capacity and a variety of functions, is a group of intracytoplasmic acidic protein. They play their unique role mainly depend in the combination with other proteins. Many studies showed that the14-3-3proteins were closely related to the occurrence and development of many kinds of tumors. It involved in tumor development mainly by regulating tumor cell growth, proliferation, migration, cell cycle, apoptosis and the signal transduction pathways. As the continuous development of life science researches, people gradually became aware of the proteins as the important executor and regulator in the life activities, they play their functions rely on the protein-protein interaction. Therefore, based on previous studies, we applied the Co-IP and3D immunofluorescence co-localization to verify that14-3-3εinteract with RKIP in gastric cancer cells and to study the mechanism which the interaction between these two proteins contribute to the proliferation and invasion in gastric cancer cells. We have examined the molecular mechanism that the14-3-3 protein could active the development of gastric cancer and provided a theoretical basis for the early diagnosis, prognosis monitoring and the targeting gene therapy of gastric cancer.The first chapter:The mechanism research of protein interaction between14-3-3ε and RKIP in gastric cancerObjective:To verify the interaction between14-3-3ε and RKIP, and explore the mechanism of their interaction to affect the cell proliferation, migration and invasion ability of gastric cancer. Methods:①Build the SGC7901gastric cancer cell lines which stable expressing RKIP and (or)14-3-3ε proteins;②Use the Co-IP and3D immunofluorescence co-localization to verify that14-3-3εinteract with RKIP in gastric cancer cells;③Apply the MTT to verify the proliferative capacity and the transwell test to verify the migration, invasive capacity of the cancer cells in each group; Explore the interaction between RKIP and14-3-3ε which could impact on the development of gastric cancer;④The protein expression levels of p-ERK and total ERK, p-RKIP and total RKIP in the cells of every groups. Results:①Build the SGC7901gastric cancer cell lines which stable expressing RKIP and (or)14-3-3ε proteins successfully;②Detected14-3-3ε protein expression in RKIP immune complex, and RKIP protein could also be detected in14-3-3ε immune complex;③RKIP and14-3-3ε proteins located in the cell membrane and cytoplasm. Two of them existed significant co-localization relationship;④Up-regulated RKIP expression levels could inhibit the proliferation, migration and invasion ability in gastric cancer SGC7901cells, and14-3-3ε played an opposite role;⑤Up-regulated14-3-3ε expression levels could increase the levels of p-ERK in SGC7901cells while up-regulated RKIP expression could decrease the p-ERK expression (P>0.05); The expression of p-ERK in between in the Co-transfected group; RKIP and14-3-3ε did not affect the levels of total ERK;⑥Up-regulated14-3-3ε expression levels could increase the levels of p-RKIP in SGC7901cells (P>0.05), but it did not affect the levels of total RKIP; RKIP was increased obviously in RKIP-transfected group.Conclusion:There is a direct or indirect interaction between RKIP and14-3-38. RKIP could enable the ERK/MAPK signaling pathway inactivated while inhibited the ability of proliferation and invasion in SGC7901cells. And14-3-3ε played an opposite role.14-3-3εcould activate the ERK/MAPK signal transduction pathway by enhancing the expression of p-RKIP. The second chapter:the mechanism research of the role of14-3-3ε in the development of gastric cancerObjective:To explore the mechanism of14-3-3sto affect the cell proliferation, migration and invasion ability of gastric cancer. To examine14-3-3ε expression in the gastric cancer tissue and its clinical significance. Methods:①Increased the expression of14-3-3ε in gastric cancer cells, and apply MTT and colony formation assay to detected the cell proliferative capacity in each group.②Apply the transwell test to verify the migration, invasive capacity of the cancer cells in each group; Explore the impact of14-3-3εon the development of gastric cancer;③Immunohistochemistry was used to detect the expression of14-3-3ε in paraffin-embedded benign gastric mucosa, primary gastric cancer and lymph node metastasis tissue to examine14-3-3ε expression in the gastric cancer tissue and its clinical significance. Results:①Up-regulated14-3-3εexpression levels could increase the proliferation, migration and invasion ability in gastric cancer SGC7901cells;②The positive expression rate of14-3-3εin the benign gastric mucosa, primary gastric cancer and lymph node metastasis tissue were35.00%(21/60)、72.04%(67/93)、100%(30/30);③The expression of14-3-3ε existed a positive correlated with the depth of invasion, TNM stage and lymph node metastasis, but a negative correlated with the degree of rumor differentiation (P<0.05). Conclusions:14-3-3ε protein could increase the cell proliferation, migration and invasion in SGC7901gastric cancer. According to the clinical pathological significance,14-3-3ε protein closely related to the malignant biological behavior of gastric cancer. It promises to be a potential molecular markers for diagnosis of gastric cancer, prediction of the metastasis of the tumor, identification of tumor differentiation, invasion depth and the TNM stage.