Construction of shRNA Expression Vector Targeting the HSP27 Gene and their Expression in Human Gemcitabine-Resistant Pancreatic Cell Line

Objective: To construct eukaryotic expression vectors of small hairpin RNA(shRNA) for HSP27 gene and investigate their expression in human gemcitabine-resistant pancreatic cells(SW1990/Gem).Methods: One pair of HSP27 shRNA sequence was selected and ligated to the pRNAT-U6.3 vector contained GFP gene and U6 promoter.pRNAT-shHSP27 or empty vector was introduced into SW1990/Gem cells by liposome-mediated transfection respectively.The transfected SW1990/Gem cells were then confirmed by fluoroscopy.The expression of HSP27 mRNA and protein was detected by reverse transcription polymerase chain reaction(RT-PCR) and Western blot,respectively.Results: Restriction enzyme digestion and sequence analysis showed that recombinant pRNAT-shHSP27 shRNA vector was successfully constructed.The expression of GFP was observed in transfected SW1990/Gem Cells by fluorescent microscopy.The expression levels of HSP27 mRNA and protein in SW1990/Gem cells transfected with pRNAT-shHSP27 shRNA vector were significantly lower than those in SW1990/Gem cells transfected with empty or negative vector.Conclusion: Recombinant pRNAT-shHSP27 shRNA vector was successfully constructed.Its transfection can silence the expression of HSP27 gene in SW1990/Gem cells.The pRNAT-shHSP27 shRNA vector lay a foundation for further study of the role of the HSP27 gene in the pathogenesis of pancreatic cancer.