【摘要】 大约30%的乳腺癌中有表皮生长因子受体家族蛋白HER2的过表达,HER2表达水平与病人的预后以及恶性程度密切相关。RNA干涉(RNAi)是最近发展起来能特异性抑制哺乳动物细胞中基因表达的新技术。在以往鉴定的对HER2有良好RNAi效应的靶序列的基础上,构建了一系列U6和H1双启动子小干涉RNA(siRNA)表达载体,并转染HER2高表达乳腺癌SKBR3细胞定量测定了其HER2下调效应。随后,siRNA表达盒经LR重组反应被克隆入慢病毒载体中并成功包装成病毒。病毒感染SKBR3后经荧光定量PCR、蛋白印迹杂交和流式细胞仪一系列实验证明:慢病毒介导的RNAi确实能有效地下调肿瘤抗原HER2的表达,而且经慢病毒处理后细胞生长受到了抑制。为进一步阐明HER2与癌症恶化的关系以及发展新的基因治疗药物提供了新的工具。更多还原

【Abstract】 HER2, a member of epidermal growth factor receptor family proteins, is overexpressed in about 30% of human breast cancer. Increased levels of HER2 are associated with poor patient prognosis and enhanced metastasis. RNA interference (RNAi) is developed recently as a new technique which can inhibit gene expression specifically in mammalian cells. On the basis of previous study,in which two target sequences with favorable RNAi effect on HER2 were identified, a series of dual promoter siRNA-expressing vectors containing two opposing U6 and H1 promoters were constructed. After transfection of HER2-overexpressing SKBR3 breast cancer cells with the siRNA-expressing vectors, downregulation of HER2 was identified quantitatively. Subsequently, the siRNA-expressing cassettes were subcloned into lentiviral vectors by LR recombination reaction and lentivirus was prepared successfully. The results from infection of SKBR3 cells with siRNA-expressing lentivirus demonstrated that lentiviral-mediated RNAi could downregulate HER2 expression efficiently through fluorescent quantitative PCR (FQ-PCR), western blot, and FACS analysis. Furthermore, cell growth was inhibited in cell proliferation assay after treatment with siRNA lentivirus.A new tool for clarifying the function of HER2 in cancer metastasis and developing the gene therapy drug was offered.更多还原