Identification And Screening Of Proteins Mucosae Carcinogenic Process

Objectives: Gastric cancer is one of the most frequently occurrence malignancies in China and the Asian-Pacific region. The research aimed to discover novel biomarkers for early detection of human gastric adenocarcinoma(GAC) and explore possible mechanisms of GAC carcinogenesis.Methods: 1. i TRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in human gastric epithelium mucosae carcinogenic process using laser capture microdissection(LCM)-purified normal gastric mucosal gland(NGM), atypical hyperplasia(AH), gastric poorly differentiated adenocarcinoma(GPDAC), lymphaden metastasis gastric adenocarcinoma(LMGAC).2. The expression of PRSS1, HSP90α/β, TGM2, Serpin A3 and P180 proteins in various stage tissues of gastric epithelium mucosae carcinogenesis were detected by Western-blot, the results were concordant with that of the proteomics. Immunohistochemistry staining was performed to detect the expression of five proteins in tissue array including NGM, AH, GAC, LMGAC, and their ability for early detection of GAC was evaluated by receiver operating characteristic analysis.3. a stable overexpression of PRSS1 in GES-1 cell line(GES-1/ pc DNA3.1-PRSS1) and a stable silencing PRSS1 expression in MGC803 cell line(MGC803/pc DNA6.2-GW/Em GFP-mi R-PRSS1) were established, and then measured the ability of cellular proliferation, apoptosis, cell cycle, immigration and invasion of PRSS1 overexpression in GES-1 cell line and MGC803 cell line of silencing PRSS1 expression by drawing cellular growth curve, colony formation, soft agar formation, flow cytometry(FCM), hoechst 33258 staining, scratch healing, Transwell migration and invasion chamber. Last, the Wnt1/β-catenin signaling way was detected by Western-blot in GES-1 cell line of PRSS1 overexpression and MGC803 cell line of silencing PRSS1 expression.Results: 1. 243 differentially expressed proteins were identified. 153 proteins were up-regulation, 91 down-regulated in GC tissue. Some proteins are progressively upregulation, some proteins are progressively downregulation in human gastric epithelium mucosae carcinogenic process, and the other proteins are up-regulation or down-regulation in a certain stage of the process of carcinogenesis.To get more insight on the biological significance of the differentially expressed proteins in gastric epithelium mucosae carcinogenic process, hierarchical clustering was performed on 243 differentially expressed proteins. All differentially expressed proteins were grouped into three groups and eight clusters. GO analysis showed that the first group was dominated by proteins associated with cellular growth, apoptosis, translation and metabolism, the second group proteins related to regulation of apoptosis, allelotaxis, and response to biotic stimulus, the third group proteins were enriched with cellular proliferation, metabolism, apoptosis, protein transport, localization and humoral immune response. KEGG pathway analysis revealed that the proteins in three groups were involved in cancer-associated signaling pathways such as cellular apoptosis, cell cycle, MAPK, p53 and Erb B signaling pathways.2. The results showed that the combined detection of PRSS1, HSP90α/β, TGM2, Serpin A3 and P180 proteins could perfectly discriminate NGM, AH, GAC and LMGAC, achieving a sensitivity of 88 % and a specificity of 84 % in discriminating NGM from AH, a sensitivity of 86 % and a specificity of 85 % in discriminating NGM from GAC, a sensitivity of 89 % and a specificity of 87 % in discriminating NGM from LMGAC, a sensitivity of 88% and a specificity of 84% in discriminating AH from GAC, and a sensitivity of 87% and a specificity of 83% in discriminating AH from LMGAC.3. The cell growth was higher, the number of plate colonies and soft agar colonies was increased, the rate of apoptosis obviously reduced, and the ability of migration and invasion was increased in GES-1/pc DNA3.1-PRSS1 cell line than in GES-1/pc DNA3.1 cell line. Moreover, the cell growth was lower, the number of plate colonies and soft agar colonies was decreased, the rate of apoptosis was added, and the ability of migration and invasion was decreased in MGC803/pc DNA6.2-GW/Em GFP-mi R-PRSS1 cell line than in MGC803/pc DNA6.2-GW/Em GFP-mi R cell line. The expression of Wnt1 and β-catenin proteins increased, but the expression of p-β-catenin protein decreased in GES-1 cell line of PRSS1 overexpression(GES-1/pc DNA3.1-PRSS1). However, it was lower that the expression of Wnt1 and β-catenin proteins in MGC803 cell line of silencing PRSS1 expression(MGC803 /pc DNA6.2-GW/Em GFP-mi R-PRSS1) than in the control cells(MGC803/pc DNA6.2-GW/Em GFP-mi R), but the expression of p-β-catenin protein increased. The expression of PRSS1 protein increased the ability of cellular growth, proliferation, migration and invasion, increased the cellular proliferation index, decreased the apoptosis rate, and the Wnt1/β-catenin signaling way was activated. Reduction of PRSS1 decreased ability of cellular growth, proliferation, migration and invasion, decreased the cellular proliferation index, increased the apoptosis rate, and the Wnt1/β-catenin signaling way was inhibited.Conclusions: 1. The research identified 243 differentially expressed proteins and studied their biological functions and signaling pathway which were related to gastric epithelium mucosae carcinogenesis.2. The results demonstrated that the combined detection of PRSS1, HSP90α/β, TGM2, Serpin A3 and P180 could perfectly discriminate NGM, AH, GAC and LMGAC. They are novel potential biomarkers for diagnosis of GAC.3. PRSS1 involved in human gastric epithelium mucosae carcinogenesis and activated Wnt1/β-catenin signaling way to affect cellular bionomic properties.