25B基因在胰腺癌细胞株中的表达及真核表达载体pcDNA-CDC25B3的构建与转染

Objective To investigate the expression of each spicing variant of CDC25B in four pancreatic cancer cell lines and construct recombinant plasmid pcDNA-CDC25B and transfect it for the further study regarding to the function of CDC25B in the development of pancreatic caner. Methods RT-PCR and Western blot are employed to assess the expression of three spicing variants mRNA and protein of CDC25B respectively. The encoding region of CDC25B3 was sub-cloned into eukaryotic expression vector pcDNA3.1(+). After verification by DNA sequencing, the acquired recombinant plasmid pcDNA-CDC25B3 was transfected into pancreatic cancer cell line SW-1990 via liposome-mediated approach in which only low expression of CDC25B protein can be detected. Selected by G418, the transfected SW-1990 was subjected to RT-PCR and Western blot assay. Results The results of RT-PCR suggested that the disparity in expression of CDC25B1 and CDC25B2 is not significant in Panc-1; the expression of CDC25B2 is higher than CDC25B1 in CFPAC-1 and SW-1990; the expression of CDC25B1 is higher than CDC25B2 in Bxpc-3; while the expression of CDC25B3 is barely detectable in four cell lines. The expression of CDC25B protein was found to be over-expressed in Panc-1 and CFPAC-1 by Western blot; medium expression of CDC25B protein was found in SW-1990; yet less to none expression could be detected in Bxpc-3. Confirmed by DNA sequencing, the recombinant vector covered the entire CDS region of CDC25B3. The transfected SW-1990 showed stable over-expression of CDC25B3 protein. Conclusion CDC25B1 and CDC25B2 are over-expressed in pancreatic cancer cells, while the relation between CDC25B3 and pancreatic caner is still not clear. We successfully constructed a pancreatic cancer cell lines with stable over-expression of CDC25B3 protein which built a foundation for clarifying the contribution made by CDC25B in the development of pancreatic cancer.