Regulation of shRNA adenovirus construction by tumor specific promoter Survivin and inhibitory effect of Survivin on expression of CD133 gene in HepG-2 cells

Abstract:

Objective：To construct small hairpin RNA driven by tumor specific promoter Survivin and to discuss the effects of down-regulating CD133 expression in HepG-2 cells line on proliferative and invasive ability of hepatoma cell. Methods：①pEntr-Sur-shRNA eukaryotic expression vector was built and HepG-2 cells，Hela cells and 293T cells were transfected. ②shRNA interference fragments were designed and CD133 interference fragments with the best effect were screened out. Carriers were reconstructed；Ad.Surp-shRNA972，Ad.Surp-shRNA485，Ad.Surp-shRNA2377 adenovirus were packed；stable expression of shRNA transfection cloning was screened out 972，2377，485 experimental groups were established by transfecting HepG-2 cells with Ad.Surp-shRNA972，Ad.Surp-shRNA2377，Ad.Surp-shRNA485；untransfected group was taken as blank control group RT-PCR and Western blot were used to test CD133 mRNA and protein expression；MTT and Transwell method were used to detect the proliferative and invasive ability of cell in vitro. Results：①Survivin promoter regulated shRNA eukaryotic expression vector of pEntr-Sur-shRNA was successful con－structed. Fluorescence was seen in Hela and HepG-2 cells but not in 293T cells indicating tumor specificity of Survivin promoter. ②Adenovirus of Ad.Surp-shRNA972，Ad.Surp-shRNA2377 and Ad.Surp-shRNA485 were successfully packaged. ③According to results of RT-PCR and Western blot，mRNA and protein levels were inhibited significantly by Ad.Surp-shRNA972 and Ad.Surp-shRNA2377 mRNA inhibition rates were（63.44±0.02）% and （56.98±0.03）% in experiment group and blank group and protein inhibition rates were （55.43±0.40）%，（48.65±0.20）%，（34.67±0.4０）% in Ad.Surp-shRNA972 group，Ad.Surp-shRNA2377 group and Ad.Surp-shRNA485 group. Cell proliferative and invasive abilities of cells in experiment group were inhibited with inhibi－tion rates of （72.88±0.74）%，（59.9±0.87）% and （30.18±0.95）% in Ad.Surp-shRNA972 group，Ad.Surp-shRNA2377 group and Ad.Surp-shRNA485 group. Conclusions：Tumor specific promoter Survivin regulating adenovirus mediated shRNA can decrease CD133 suppression and inhibit proliferation and invasiveness of HepG-2 hepatoma cell.