肿瘤特异性启动子Survivin调控shRNA腺病毒的构建及对HepG-2细胞中CD133基因表达的抑制

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

摘要:

目的：构建肿瘤特异性启动子Survivin驱动的小发夹RNA（shRNA），通过下调HepG-2肝癌细胞中CD133的表达，探讨其对肝癌细胞增殖和侵袭能力的影响。方法：①构建pEntr-Surp-shRNA真核表达载体，转染HepG-2肝癌细胞，Hela细胞，293T细胞。②设计shRNA干扰片段，筛选干扰最佳干扰效率的CD133干扰片段，重组载体pAd.Surp-shRNA972，pAd.Surp-shRNA2377，pAd.Surp-shRNA485并筛选出稳定表达shRNA的转染克隆。③Ad.Surp-shRNA972，Ad.Surp-shRNA2377，Ad.Surp-shRNA485腺病毒分别转染HepG-2肝癌细胞株，建立Ad.Surp-shRNA972，Ad.Surp-shRNA2377，Ad.Surp-shRNA485实验组，未转染腺病毒组为空白组。感染肝癌细胞株，应用RT-PCR和Western blot检测CD133 mRNA和蛋白的表达，MTT法和 Tran－swell 法检测细胞体外增殖和侵袭能力。结果：①成功构建了Survivin 启动子调控的shRNA真核表达载体pEntr-Surp-shRNA，在293T细胞株无荧光，而在Hela、HepG-2细胞株中荧光表达明显，证实Survivin具有肿瘤特异性。②成功包装Ad.Surp-shRNA972，Ad.Surp-shRNA2377，Ad.Surp-shRNA485腺病毒。③RT-PCR和Western blot检别显示Ad.Surp-shRNA972、Ad.Surp-shRNA2377，有封闭CD133 mRNA和蛋白表达的效果；计算实验组与空白组相比mRNA抑制率分别为（63.44±0.02）%、（56.98±0.03）%；Ad.Surp-shRNA972、Ad.Surp-shRNA2377、Ad.Surp-shRNA485组蛋白抑制率分别为（55.43±0.40）%、（48.65±0.20）%、（34.67±0.40）%；实验组细胞的增殖和侵袭能力也受到明显抑制，Ad.Surp-shRNA972组生长抑制率为（72.88±0.74）%，Ad.Surp-shRNA2377组生长抑制率为（59.90±0.87）%，Ad.Surp-shRNA485组生长抑制率为（30.18±0.95）%。结论：肿瘤特异性启动子Survivin调控腺病毒介导的shRNA能显著下调CD133基因的表达，抑制肝癌细胞株HepG-2的增值和减弱其侵袭力。

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.