| Objective:Pancreatic cancer is a highly malignant tumor of digestive system ,and it is seriously harm to human's life and health. Because of its hidden disease and rapid progress, the effect of traditional treatment is not satisfactory.Its treatment is very difficult and prognosis is very poor, so it is very important to developed a new method of diagnosis and treatment of pancreatic cancer. In recent years, with the rapid development of gene therapy and genetic research of pancreatic cancer, gene diagnosis and gene therapy of pancreatic cancer are being further explored.The blood vessel growth of pancreatic cancer plays a very important role in the development and metastasis of tumor,so it is very important to inhibit the growth of pancreatic blood vessels and prevent rapid growth of the tumor. EphB4 receptor promotes the sprouting,migration,proliferation and tube formation of endothelial cell,and regulates arterial and venous differentiation during embryonic angiogenesis.In the present,it has been reported that high expression of EphB4 receptor in pancreatic ductal cell carcinoma is closely related to tumor angiogenesis and invasion.RNA interference (RNAi)is a post-transcriptional gene silencing, it can trigger post-transcriptional control procedures and lead to degradation of specific single- stranded mRNA.RNAi has been successfully used in gene function and upstream and downstream molecular interactions of signal transduction system,and provide a new strategy for cancer therapy.In this study, eukaryotic expression plasmid vector of targeting EphB4 was constructed and transfected to PANC-1 pancreatic cancer cells to research the inhibition of RNAi for PANC-1 cells on gene expression of EphB4,cell growth and migration.It provides the basis for gene therapy of pancreatic cancer in the future.Methods:①software analyzed mRNA of EphB4 gene and selected target sites.Constructing EphB4 recombinant eukaryotic expression plasmid pSIREN-RetroQ- ZsGreen-EphB4 and constructing a negative control plasmid pSIREN-RetroQ- ZsGreen-N.After sequencing, the recombinant plasmid was extracted and transfected to PANC-1 cells;②The experiment was divided into control group, pSIREN-RetroQ- ZsGreen-EphB4 group and pSIREN-RetroQ-ZsGreen-N group for the vitro experiments;③Transfecting plasmid to cells after 48h,the relative amount of EphB4 mRNA and protein were measured by RT-PCR and Western-blotting,and observeing the inhibition of the recombinant plasmids for the expression of EphB4 gene of PANC-1 cells;④Cell proliferation was determinated by MTT;⑤Scratch migration assay detected the ability of cell migration.Results:①sequencing proved that the recombinant plasmid pSIREN-RetroQ- ZsGreen-EphB4 and pSIREN-RetroQ-ZsGreen-N were successfully constructed;②Transient transfecting after 48h, the expression level of EphB4 mRNA in the group of liposomes and interfere plasmid more decreased than the control group, the group of liposome, the group of interfere plasmid group, and the group of control plasmid liposome (P <0.05) . There were not significant difference between the control group, the group of liposome,the group of interference plasmid and the group of control plasmid liposome (P> 0.05);③Western-blot method showed that the expression level of EphB4 mRNA in the group of liposome interference plasmid more decreased than the control group and the group of control plasmid liposome (P <0.05). There were not significant difference between the blank group and the group of control plasmid liposome (P> 0.05);④the inhibition of EphB4 expression weakened the proliferation of PANC-1,and the ability of cell migration compared with the control group decreased.Conclusion: The siRNA eukaryotic expression plasmid vector of targeting EphB4 was successful constructed and effectively transfected PANC-1 pancreatic cancer cells.Recombinant plasmid can inhibit the expression of EphB4 gene mRNA and protein of pancreatic cancer PANC-1 cells ,and the growth and migration.of PANC-1 cells were significantly decreased. |
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