摘要
目的应用RNA干扰技术研究E2F-1基因沉默在人胃癌MGC803细胞中对Rb蛋白表达的影响。方法将重组质粒E2F-1-siRNA转染MGC803细胞,筛选稳定株,利用RT-PCR检测转染前后细胞E2F-1mRNA表达水平,并通过蛋白免疫印记法(Western blot)检测各组细胞E2F-1蛋白的表达水平来验证质粒转入胃癌细胞的情况。采用Western blot检测三组细胞中总Rb蛋白和磷酸化Rb蛋白的表达情况,计算出非磷酸化Rb蛋白表达情况,统计各组间的差异。结果重组质粒E2F-1-siRNA成功转入胃癌细胞中;有效抑制了E2F-1 mRNA的表达,E2F-1蛋白水平显著下降,与阴性对照组及未转染组相比分别降低了83.2%和84.6%,去磷酸化Rb蛋白分别增加了61.22%(P<0.05)和66.60%(P<0.05),差异有统计学意义。结论沉默表达E2F-1基因后,可以有效降低Rb蛋白的水平,促进其活化形式去磷酸化Rb蛋白的产生。
Objective To investigate the effect of siRNA-mediated gene silencing on the expression of Rb gene protein in MGC803 cells. Methods The siRNA expression plasmids against E2F-1 were transfected into MGC803 cells in vitro. After 48 hours, E2F-1 mRNA expression levels were detected by semi-quantitative RT-PCR and western blot analysis. The levels of total Rb protein and Rio phosphorylation protein expression were also analyzed by western blot. And the expression of the dephosphorylation of Rb could be calculated from these results above. Re- suits Expression of plasmid DNA was stable after transfection into MGC803 cells and the mRNA level of E2F-1 in MGC803 cells was downregulated significantly comparing with normal group and negative control group. Western-blot analysis has shown that the levels of the expression of E2F-1 protein was downregulated in MGC803 ceils after interfered with siRNA （83.2%, 84. 6%, P 〈0. 05 ） while the expression of Rb dephosphorylation protein was upregulated （61.22%, 66. 60%, P 〈 0. 05）. Conclusion The mRNA and protein level of E2F-1 can be downregulated by using RNA interference in MGC803 ceils, and therefore siRNA-mediated E2F-1 upregulate the activity of Rb phosphorylation protein. There might be some antagonistic effects between E2F-1 gene and Rb gene. So E2F-1 might be used in clinical gene therapy for gastric carcinoma.
出处
《中国临床新医学》
2012年第4期281-284,共4页
CHINESE JOURNAL OF NEW CLINICAL MEDICINE
基金
国家自然科学基金资助项目(编号:30860273)
广西科学研究与技术开发计划项目(编号:桂科攻10124001A-22)
关键词
E2F-1
RB蛋白
去磷酸化
siRNA
胃癌
E2F-1
Retinoblastoma
Dephosphorylation
RNA interference
Gastric carcinoma
