特异性siRNA质粒的构建及其抑制癌细胞中mcl-1表达的研究 The Specific siRNA Plasmids Construction and their Inhibition Effect on Expression of mcl-1 Protein in Liver Cancer Cell.pdfVIP

现代食品科技 Modern Food Science and Technology 2010, Vol.26, No.8 特异性siRNA质粒的构建及其抑制癌细胞中 mcl-1 表达的研究 徐爱群，曹以诚，区镜深，张珍武 （华南理工大学生物科学与工程学院，广东广州 510006） 摘要：本文构建了靶向 mcl-l 基因构建的特异性 siRNA 重组质粒，在细胞水平检测的其抑制效果，并筛选出能高效抑制 mcl-l 基因表达的 siRNA 重组质粒，针对mcl-1 的特异性siRNA 重组质粒，将其转染到人肝癌细胞HepG2 ，用Rt-PCR( 实时荧光定量PCR) 方法和 Western Blot 方法分别检测转染前后 mcl-1mRNA 水平和 Mcl-1 蛋白表达水平，比较对应不同位点的两个 siRNA 重组质粒对 mcl-1 的抑制效果。结果表明，Rt-PCR 结果显示对应不同位点的两个 siRNA 重组质粒对mcl-1 均可降低mcl-1 mRNA 水平，最大抑制 效率达 70.0%，远高于作为对照非相关组；Western-Blot 结果显示转染特异性 siRNA 重组质粒后Mcl-1 蛋白表达受到抑制，最大抑制 率为44.2%。合理设计的特异性siRNA 重组质粒可大幅度下调mcl-1 mRNA 水平，能有效抑制Mcl-1 蛋白表达。 关键词：肝癌；mcl-1；基因沉默；实时荧光定量PCR 文章篇号：1673-9078(2010)8-789-792 The Specific siRNA Plasmids Construction and their Inhibition Effect on Expression of mcl-1 Protein in Liver Cancer Cell XU Ai-qun, CAO Yi-cheng, OU Jing-shen, ZHANG Zhen-wu (Bioscience and Bioengineering School of South China University of Technology, Guangzhou 510006, China) Abstract: The mcl-l gene-specific siRNA plasmids were constructed and transfected into HepG2. The levels of mcl-1 mRNA and Mcl-1 protein expression were detected with the Rt-PCR (real time PCR) and WesternBlot methods, respectively. And the inhibitory effects of siRNA plasmid corresponding to two different sites on mcl-1 expression were compared. Rt-PCR results showed that both the two siRNA plasmids correspomding to two sites could reduce the levels of mcl-1 mRNA and the maximum inhibition efficiency was of 70.0%, which was much higher than that of a control group. Western-Blot results were showed that transfection of mcl-l gene-specific siRNA plasmids into HepG2. The expression of Mcl-1 protein obviously inhibit the expression of mcl-l protein with the maximum inhibition