慢病毒介导的RNA干涉对乳腺癌SKBR3细胞HER2受体的下调及生长抑制  

Silencing of HER2 Receptor and Growth Inhibition of SKBR3 Breast Cancer Cells by Lentiviral-mediated RNAi

作　　者：程联胜[1] 查昭[1] 席甲甲[1] 江冰[1] 刘兢[1] 姚雪彪[1] 

机构地区：[1]中国科学技术大学生命科学学院安徽省分子医学重点实验室,合肥230027

出　　处：《中国生物工程杂志》2007年第2期1-8,共8页China Biotechnology

基　　金：国家自然科学基金(30400078);国家973计划(2002CB713700);安徽省自然科学基金资助项目(050430201)

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。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, downregnlation 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.

关 键 词：HER2 RNA干涉 小干涉RNA 慢病毒 基因治疗 

分 类 号：Q78[生物学—分子生物学] R394[医药卫生—医学遗传学]