The Effect Of WWOX Eukaryotic Expression Vector On Human Gallbladder Carcinoma Cell Line GBC-SD In Vitro

Objective To construct eukaryotic expression vector of WWOX gene, and to investigate the effect of transfection with the WW domain-containing oxidoreductase (WWOX) gene on cell proliferation and apoptosis in human gallbladder carcinoma cell line GBC-SD.Methods Total mRNA was extracted from GBC-SD cell lines, the cDNA of anti-cancer gene WWOX was cloned by RT-PCR, the cDNA of WWOX and pcDNA3.0were cut by restriction endonuclease ECOR Ⅰ and Xho Ⅰ,then they were joined together, We gained recombined plasmid vector pcDNA3.0-WWOX containing anti-cancer gene WWOX. The human gallbladder carcinoma cell line GBC-SD was transfected by WWOX gene with liposome Lipofectamine2000, The mRNA and protein expression of WWOX in GBC-SD cells transfected with the recombinant plasmid was detected by quantitative RT-PCR and Western blot. Cell proliferation was tested by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis was assessed by flow cytometry (FCM).Results1. It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic expression vector pcDNA3.0-WWOX was correct.2. The human gallbladder carcinoma cell line GBC-SD was transfected by WWOX gene with liposome Lipofectamine2000.3. The expression of WWOX mRNA was markedly increased in GBC-SD cells transfected with the recombinant plasmid compared with control groups.4. Immunofluorescence cell staining result:The expression of WWOX protein was markedly increased in GBC-SD cells transfected with the recombinant plasmid compared with control groups.5. Western Blot result: The expression of WWOX protein was markedly increased in GBC-SD cells transfected with the recombinant plasmid compared with control groups.6. WWOX gene transfection significantly decreased GBC-SD cell proliferation compared with control groups.7. WWOX gene transfection significantly promoted GBC-SD cell apoptosis compared with control groups.Conclusion1.The eukaryotic expression vector pcDNA3.0-WWOX was successfully constructed.2. WWOX gene transfection significantly decreased GBC-SD cell proliferation but promoted apoptosis when compared with control groups.