| BackgroundPancreatic carcinoma is one of the most common malignant tumors which has high degree of malignancy and poor prognosis. The morbidity of pancreatic carcinoma is becoming higher in recent years in the worldwide. The early diagnosis and treatment of pancreatic carcinoma are very difficult because there are no evident symptoms and signals in its early stage. Beside this, the operating resecting rate and five-year survival rate of pancreatic carcinoma are really low, so it has become one of the most severe malignant diseases and threatens the health of human being.Over years, many scientists have done a lot of researches on pancreatic carcinoma from gene level. However, genes are only carriers of genetic messages, and the essences of lives are results of protein interactions. As carriers between cell and outside, cell membranes play various important roles in maintaining cellular micro-environment, mediating the energy and material transmission, signal transduction and so on. In recent years, membrane proteomics has proved to be very effective in the research of disease pathogenesis. So in our study, we used membrane proteomic techniques to screen different membrane proteins in pancreatic carcinoma cells, then analyzed and identified the biological and clinical pathological functions of target membrane proteins. We expected to figure out if this target protein participate in the pathogenesis of pancreatic carcinoma and find a potential target of early diagnosis and treatment of pancreatic carcinoma.Objective1. To establish a new proteomics system of extracting, isolating and identifying plasma membrane protein effectively.2. To screen significative membrane proteins of pancreatic carcinoma through membrane proteomics analysis of pancreatic carcinoma PANC-1cell line.3. To investigate the biological functions in pancreatic cancer cells and verify the expression levels of target membrane proteins in definite pancreatic carcinoma specimens, then to explore the roles of target membrane proteins in pancreatic carcinoma pathogenesis and provide a new thought of early diagnosis and treatment of pancreatic carcinoma.Methods1. Pancreatic carcinoma PANC-1cells were cultured in vitro. Plasma membrane proteins were extracted through modified aqueous two-phase partition system. Membrane proteins of PANC-1cell line were isolated by using ASB-14and CHAPS separately as the zwitterionic detergent and identified by using2D-MALDI-TOF-TOF-MS/MS technique. Bioinformatic softwares, such as GOA、 DAVID、STRING, were used to analyze the membrane proteins.2. Apoptosis was induced through Doxrubicine in both pancreatic carcinoma PANC-1and BxPC-3cells. After transfected with targeted siRNAs against membrane proteins, the expression levels of apoptosis-related proteins CASP3and PARP1were detected through Western blot technique.3. Tissue specimens from Qilu Hospital of Shandong University were collected, all of which had been definitely diagnosed as pancreatic carcinoma according to pathological results. The differentiation degrees of these11specimens were different. Immunohistochemistry method was used to detect the expression levels of target membrane protein in pancreatic ductal adenocarcinoma and para-carcinoma tissues.Results1. Identification of plasma membrane proteins of pancreatic carcinoma PANC-1cell lineIn this study, fifty-three proteins were isolated and identified, among which50proteins had gene ontology annotation (GOA) notes. In all53proteins, thirty-two were membrane-associated proteins. The biological function of prohibitin-1(PHB1) in pancreatic carcinoma remained unclear, so we chose PHB1to do further analysis.2. Analysis of biological function of PHB1After the PHB1levels were knocked down by targeted siRNA, PANC-1and BxPC-3cells were treated with Doxorubicin (1μM) for24hours. The results of Western blot showed that the expression of CASP3and PARP1which were related to apoptosis were down-regulated. PHB1play an important role in apoptosis process of pancreatic cancer cell.3. Analysis of different expressions of PHB1in both para-carcinoma tissue and different differential pancreatic carcinoma.Eleven specimens were collected, which had been definitely diagnosed as pancreatic ductal adenocarcinoma according to pathologic analysis. The expressions of PHB1were analyzed through immunohistochemistry technique between cancer tissues and para-cancer tissue. The results showed that expressions of PHB1were lower in differently differentiating cancer tissues than in para-cancer tissues. There was a positive correlation between the expressions of PHB1and the differentiation levels of cancer tissues.Conclusions1. Aqueous two-phase partition system combined with modified two-dimensional electrophoresis can be used to extract, isolate and identify plasma membrane proteins. This technique has high efficiency in membrane protein extraction and identification.2. Cellular function analysis showed that the expressions of apoptosis-related proteins decreased significantly in PHB1deleted cells exposed to chemotherapeutic drug, which demonstrated that PHB1may be an important regulator of apoptosis in pancreatic carcinoma. Therefore, PHB1could be a new potential target of anti-cancer drug development for pancreatic carcinoma.3. The results of immunohistochemistry analysis showed that PHB1could be used not only as one of the biomarkers to distinguish cancer tissue and para-cancer tissue, but also as a criteria of different differentiating grades of pancreatic carcinoma, which could be helpful in early diagnosis and treatment of pancreatic carcinoma.In conclusion, we provided a usefull method to isolate and identify plasma membrane proteins, and demonstrated that PHB1may be a promising biomarker for early diagnosis and therapy of cancers. |
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