引用本文:李 芳1,邹冬玲2,曹 姝1,李少林1.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[J].重庆医科大学学报,2013,(12):1419~1424
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
DOI:
中文关键词:  Survivin  CD133  小发夹RNA  肝癌
英文关键词:Survivin  CD133  shRNA  hepatoma
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作者单位
李 芳1,邹冬玲2,曹 姝1,李少林1 1. 重庆医科大学基础医学院放射医学教研室重庆 4000162. 重庆市肿瘤医院妇产肿瘤科重庆 400016 
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中文摘要:
      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。
英文摘要:
      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.
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