| Pancreatic cancer (PC) is one of most common gastrointestinal cancers with poor prognosis, due to the obsolete clinical manifestations, faster development and difficult early diagnosis. There has been such a high mortality rate of pancreatic cancer, and most diagnosed patients have lost the chance of surgery. Therefore finding and studying diagnostic markers for early stage pancreatic cancer are significant for improving the prognosis of pancreatic patients.Phosphorylated protein50(EBP50), also known as Na+/H+exchanger regulatory factor1(NHERF1), are comprised of six multifunctional exons with358amino acids. The EBP50gene localizes at17q25.2, which was identified to be a suppressor gene in various tumors recent years. Previous research mainly focused on the significance and mechanisms of EBP50expression in human breast and liver cancers. However, up to date, the expression and function of EBP50in the malignant progression of pancreatic cancer have not been described. Therefore, further study is needed to explain the roles and mechanisms of EBP50involved in pancreatic cancer malignant progression.Part1:The significance of EBP50expression in pancreatic cancer using the quantum dots assayPurpose:To analyze the significance of EBP50expression in pancreatic cancer, the quantum dots assays were performed to detect the expression levels of EBP50in normal pancreatic, L-PanIN, H-PanIN and pancreatic cancer tissues, and analyze the relationship between EBP50expressions with the clinical pathologic characters in pancreatic cancer tissues.Methods:The quantum dots immunohischemical (QD-IHC) was used to examine EBP50expression in the40samples with normal pancreatic tissues,80samples with pancreatic cancer tissues,40samples with L-PanIN tissues and40samples with H-PanIN tissues. We analyzed the differences of EBP50expression between the four groups. The expression of EBP50in cancer tissues associated with patient clinical data were also analyzed including sex, age, differentiation, TNM classification, lymph node metastasis, tumor area and tumor size.Result:The total EBP50expression intensity was compared by densitometry. The relative values for NP, L-PanIN, H-PanIN and PC were67.34±2.69,65.51±1.92,70.13±2.61, and36.81±1.22respectively. The H-PanIN tissues showed the highest EBP50expression (P<0.05), while pancreatic cancer presented the lowest EBP50expression (P<0.05). The present study indicates that the EBP50expression pattern changes during transformation, as there is a loss of the normal apical membrane distribution and an ectopic cytoplasmic over-expression of EBP50; furthermore, the EBP50level is subsequently decreased during malignant progression of PC. In addition, EBP50expression in PC tissues was significantly associated with TMN staging, differentiation level and lymph node metastasis (P<0.05), but not with sex, tumor area, tumor size and age (P>0.05).Conclusion:Pancreatic cancer tissues showed significantly lower EBP50expression levels. And sub-localization of EBP50changed during malignant progression of PC. EBP50expression was significantly associated with pancreatic clinicopathological prognostic factors, indicating that EBP50may be a promising target to assess the pancreatic malignancy, early diagnosis and prognosis.Part2:The effects of up-regulating and down-regulating EBP50expression on pancreatic cancer cells in vitroPurpose:To study the effects of up-regulating and down-regulating on the proliferation of pancreatic cancer cells, furthermore explain the potential mechanisms of EBP50involved in pancreatic cancer malignant progression.Method:The EBP50plasmid and EBP50siRNA was transfected into PC cell line, and western blot was used to detect the EBP50expression in transfected cells and controls. The proliferation rate of six cell lines was measured by CCK-8assay. The colony-forming ability of six cell lines was assayed by soft agar colony formation assay. What’s more, the protein levels of β-catenin, pRb, P27and cyclin E were measured by western blot.Results:Western blot showed that stable transfected cell lines were successfully constructed. Up-regulation of EBP50expression significantly inhibited the growth, the colony-forming ability of cells and arrested the G1-to-S progression. Additionally, the over-expression of EBP50attenuated β-catenin activity, decreased cyclin E and phosphorylated Rb expression, and accentuated p27expression compared to control cells. This finding was further strengthened by repressing EBP50expression by siRNA that exerted the opposite effects in PC-2cells.Conclusions:EBP50may inhibit proliferation in human pancreatic cancer cells through regulating the Wnt/p-catenin pathway and cell cycle-related proteins, and EBP50shows significant anti-cancer effects in vitro, indicating that EBP50may function as a potential tumor suppressor in PC.Part3:The effects of up-regulating and down-regulating EBP50expression on pancreatic cancer in vivoPurpose:To investigate the effects of EBP50over-expression and down-regulation on the growth of transplantation tumor in human pancreatic cancer cell lines.Methods:The BALB/C nude mice were divided into six groups, and were inoculated by PANC-1cells, HA-PANC-1cells, EBP50-PANC-1cells, PC-2, NTCsiRNA-PC-2and EBP50siRNA-PC-2, respectively. The status of nude mice and tumor growth were observed. After30days, the nude mice were sacrificed, and the volumes and quality of tumors were obtained for statistical analyses. In addition, immunohistochemitry assay was performed to detect EBP50expression in dislodged tumors.Results:The tumors successfully presented and grew after inoculation of six cancer cell lines, in the subcutaneous of nude mice. The volumes and mass of EBP50-PANC-1group tumor respectively were (1300±212)mm3and (0.6±0.08) g, which were significantly less than PANC-1group (2200±236) mm3,(1.2±0.16) and HA-PANC-1group (2050±222) mm3,(1.1±0.18) g (P<0.05). And the volumes and mass of EBP50siRNA-PC-2group tumor respectively were (2750±180)mm3and (1.5±0.12) g, which were significantly more than PC-2group (2100±140) mm,(1.1±0.18) g and NTCsiRNA-PC-2group (2230±100) mm3,(1.2±0.07)g (P<0.05). The immunohistochemitry staining showed that the EBP50expression level in EBP50-PANC-1induced tumors was obvious higher than that in the two control groups, revealing that EBP50was also over-expression in EBP50-PANC-1cells after injecting into the subcutaneous of nude mice for30days. In EBP50down-regulation group, the EBP50expression level in EBP50siRNA-PC-2induced tumors was obvious less than that in the two control groups.Conclusion:EBP50over-expression markedly inhibits the growth of tumor, and down-regulation of EBP50expression accerlerates the growth of tumor, indicating that EBP50might be developed as a novel therapeutic intervention for pancreatic cancer in... |
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