Screening And Identification Of Differentiation-related Proteins In Gastric Cancer Tissues

Objective: Gastric cancer is the most common malignant tumor of thedigestive system. The incidence rate of gastric cancer is the second highestand the mortality rate of gastric cancer is the third highest in a variety ofmalignant tumors among the population. While comprehensive treatment forpatients is mainly based on surgery, five-year survival rates of them have notbeen significantly improved. Studies have shown that the degree of tumordifferentiation is closely related to the survival rate of patients, especially inpoorly differentiation gastric cancer of patients with a worse prognosis. Nowproteomics research between gastric cancer tissue and normal tissue are morethan that of different differentiated gastric cancer. So it is very important forus to find out and filter the specific different proteins between differentdifferentiated gastric cancer so as to assess the degree of gastric cancer and toanalyze the prognosis. This research screens the different proteins in the welland poorly differentiated tissues of gastric cancer by SDS-PAGE and LC-MC.We search differences in proteins and then identify them, in order to find theregulatory proteins closely related to the differentiation of tumors. Theyprovide the theoretical and experimental basis for the differentiationmechanism of gastric cancer and reversal research. Then the intervention ofcertain genes of signaling pathways can achieve inhibition of tumor celldifferentiation or even reverse.Methods:1We collect the gastric cancer tissues, select eight cases of welldifferentiated gastric cancer tissues and eight cases of poorly differentiatedgastric cancer tissues by pathological diagnosis and extract total solubleprotein from these tissues and test the concentration of protein samples usingthe Bradford method. 2We separate the proteins by SDS-PAGE and then compare the gelelectrophoresis of the well and poorly differentiated gastric cancer tissues soas to find and excise the different protein bands.3We dissolve the different protein bands filtered in the gel, analyze thedifferentially expressed proteins extracted by LC-MS and then identify theprotein by biological information database.4We select and verify three of the eight differentially expressed proteinswhich were identified by QPCR and Western blot on the mRNA and proteinexpression levels in order to test whether it maintains consistency with theproteomics test results.Results:1We get the gel map of well differentiated gastric cancer and poorlydifferentiated gastric cancer by SDS-PAGE. We get15different protein bandsby comparison, cut and dissolve them and then identify355proteins byLC-MS.BY screening the high abundance and structural protein, we determineeight proteins that may be related to differentiation-related protein of tumor.They are Cofilin, proteaseactivation subunits(PSME1),Ras-related protein7a(RAB7a), filamin A (FLNa), pyruvate kinase (PK),glutathione S-transferaseenzyme-Ï€-1(GST-Ï€-1),the peroxidase5(PRDX5), actin-related protein.2We verify three of those important differentially expressed proteinswhich are Cofilin RAB7a and PSME1in the mRNA levels. The result ofReal-time PCR shows that these three kinds of proteins are significantlydifferent in well differentiated and poorly differentiated gastric cancer tissues(t=-2.600,t=-5.285,t=4.279;P<0.05), which is consistent with the proteomicsresult.3Then we verify three differentially expressed proteins in the mRNAlevels. The result of western blot shows that Cofilin and RAB7a aresignificantly reduced (t=-13.223,t=-21.579;P<0.01),while PSME1issignificantly enhanced(t=9.991;P<0.05), compared with poorly differentiatedgastric cancer, which is also consistent with the proteomic result. Conlusions:1We find eight differentially expressed proteins in differentdifferentiated gastric cancer tissues. Protein expression is confirmeddifferently in the various degrees of differentiation of human gastricadenocarcinoma. These differentially expressed proteins are scattered in thecytoplasmic proteins, nuclear proteins, cytoskeletal proteins and intracellularorganelle protein. They play important roles in gastric cancer cell signaltransduction, maintenance of cell morphology and normal defense functionand the promotion of cell division, proliferation and apoptosis.2Three of them(Cofilin, RAB7a and PSME1)are verified by QPCR andWestern blot on the mRNA and protein expression levels. The results areconsistent with the proteomics results, and this confirms that the proteinexpressions are different in various degrees of differentiation of human gastriccancer tissues.3The study of the function of differentially expressed proteins cancontribute to the study of biological behavior of gastric cancer differentiation,provide a theoretical basis for clarifying the different differentiated gastricadenocarcinoma in the development of molecular mechanisms andphysiological and pathological systems, and also give a new target for tumorbiological therapy.4We build the map of the well and poorly differentiated human gastricadenocarcinoma, and identify eight differentially expressed proteins. We findout the closely related proteins with tumor differentiation, which providetheoretical and experimental basis for tumor gastric cancer differentiationmechanism and study of reversal.