The Effects And Mechanism Of Up-Regulating The EBP50 Expression On Pancreatic Cancer Cells SW1990

Pancreatic cancer (PC) is one of the most common malignant gastrointestinal cancers with high mortality rate and poor prognosis. Patients who were definitly diagnosised almost were performed as advanced PC even lost opportunity of operation for reson as accompanied by lymph node or distant metastasis. Lack of specificity clinical manifestations and effective early diagnostic methods were key problems. The pathogenesis of pancreatic cancer is not entirely clear yet, oncogene activation, anti oncogene dysfunction and signal transduction pathway abnormalities were thought to be involved in the occurrence of cancer. Therefore, studying the pathogenesis of pancreatic cancer, looking for early effective tumor marker for diagnosis and prognostic significance is of great significance.Phosphorylated protein 50 (EBP50) is also known as sodium hydrogen exchanger regulating factor 1 (NHERF1), was discovered in recent years as a widely tumor related anti oncogene, such as breast cancer, liver cancer, colorectal cancer, gastric cancer. This study detected EBP50 in pancreatic cancer tissue and upregulate the expression of EBP50 to study the effect on pancreatic cancer cell lines SW1990 and specific mechanisms, with the purpose of providing new evidence to the diagnosis and treatment of pancreatic cancer.Part I To detect the EBP50 expression in pancreatic cancer using Immunohistochemistry and RT-qPCRObjectives:To detect and compare the levels of EBP50 expresses in pancreatic cancer and normal pancreatic.Method:The Immunohistochemistry(IHC) was used to examine EBP50 expression in 120 pancreatic cancer tissue samples and the RT-qPCR was used to detect the mRNA level in 20 cancer tissue samples and 20 noncancer samples.Results:The IHC results show that:of 120 PC tissue samples,45(37.5%) cases had no EBP50 expression,38 (31.67%) were dyed 1+,22(18.33%) were staing 2+and only 15(12.5%) were postive 3+. The normol tissule always were postive 3+. The RT-qPCR show that the mRNA level of 20 cancer tissue samples was 0.75±0.11 while the noncancer tissuse was 1.63±0.19.Conclusion:Pancreatic cancer tissues show significantly lower EBP50 expression levels and it was an significantly pancreatic clinicopatholoogical prognostic factorsPart ? The effects of up-regulating the EBP50 expression on pancreatic cancer cells SW1990Purpose:To study the effects of up-regulating the EBP50 expression on the proliferation and invasion of pancreatic cancer.Method:SW1990 cells were cultivate with DMEM-F12 culture of 10% Fetal bovine serum, then build stable transfection cells mediate by lipidosome and screen with G418, the western blot were performed to detect the expression of EBP50 in transfected and controls. The CCK-8 assay and the soft agar colony formation assay were apartly used to detect the proliferation and colony-forming ability of three cell lines. The invasive ability was detect by Transwell.Results:EBP50-SW1990 cell and HA-SW1990 cell were successfully build and Western blotting showed that EBP50 expression was significantly highly in EBP50-SW1990 cell compared with HA-SW1990 cell and SW1990 cell the different was signifiant(P<0.05). Compared with two control cells, EBP50-SW1990 cell showd less proliferation and colony-forming ability with significant different(p<0.05). Transwell candidate the invasive ability of EBP50-SW1990 was also decreased.Conclusion:The EBP50 over-expression inhibite proliferation and invasion of the SW1990 cell.Part III The mechanisms of up-regulating the EBP50 expression on pancreatic cancer cells SW1990Purpose:To explain the potential mechanisms of EBP50 involved in malignant progression of pancreatic cancer.Method:The cycle kit was used to detect the cell cycle of EBP50-SW1990 cell, HA-SW1990 cell and SW1990 cell and apoptosis conditions of three cell lines were measured by Hoechst 33258. The protein level of Bcl-2, ?-catenin and E- cadherin were mesured by western blotting.Results:Compare to HA-SW1990 cell and SW1990 cell, the G1/G0 cells of EBP50-SW1990 cell were significantly increased(62.7%±1.03%) while the S stage cells were reduced(15.3%±1.33%). The Hoechst 33258 also result that EBP50-SW1990 with significantly high apotosis ratio(P<0.05). The expression level of Bcl-2 was decreased in EBP50-SW1990 cell and it also showed down-expression of E-cadherin and up-expresion of ?-catenin.Conclusion:Up-regulation of EBP50 expression significantly arrested the G1-to-S progression and induced apoptosis. Additionally, the over-expression of EBP50 regulate the PC through accentuated E-cadherin activity, attenuated ?-catenin and Bcl-2 expression. EBP50 may function as a potential tumor suppressor.