腺病毒介导的RNA干涉对乳腺癌SKBR3细胞HER2的下调及生长抑制效应  

Downregulation of HER2 by adenovirus-mediated RNA interference and its inhibitory effect on growth of SKBR3 breast cancer cell

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细胞与分子免疫学实验室安徽省分子医学重点实验室,安徽合肥230027

出　　处：《细胞与分子免疫学杂志》2007年第8期691-695,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30400078);国家重点基础研究发展规划(973)项目(2002CB713700);安徽省自然科学基金资助课题(050430201)

摘　　要：目的:探讨采用腺病毒做为载体介导基于RNA干涉的针对高HER2肿瘤的基因治疗的可能性。方法:构建HER2-绿色荧光蛋白融合蛋白表达质粒pHER2-GFP,并与9种针对HER2不同靶序列的siRNA表达质粒分别共转染CHO-K1细胞,根据荧光蛋白表达量从中筛选出沉默效率最高的质粒。将筛选出的质粒转染HER2高表达乳腺癌SKBR3细胞,检测它们对HER2表达的影响。随后,将siRNA转录单元克隆入腺病毒载体,成功包装病毒后感染SKBR3细胞,再次测定其下调HER2的效应及其对细胞生长的影响。结果:2种有效下调HER2表达的质粒被筛选出来。将此2种质粒所含siRNA转录单元包装入腺病毒后仍然保持了原有的基因沉默效应。HER2下调增加了SKBR3细胞中G1期细胞的比例,并且诱导部分细胞凋亡。MTT和细胞长期增殖抑制实验表明腺病毒介导的RNA干涉抑制了SKBR3细胞生长。结论:重组腺病毒介导的RNA干涉能够下调HER2的表达并且对高表达HER2的乳腺癌细胞有生长抑制作用。AIM： To explore the possibility of RNA interference （RNAJ）-based gene therapy against HER2-overexpressing tumors using adenovirus-mediated vector. METHODS： A plasmid named pHER2-GFP containing HER2 and green fluorescent protein （GFP） fusion was constructed and co- transfected into CHO-K1 cells respectively with nine small in- terference RNA （siRNA）-expressing plasmids targeting dif- ferent regions of HER2. The siRNA-expressing plasmids with best interference effect were screened out and then used to identify the gene silence effect in HER2-overexpressing SKBR3 breast cancer cells. Subsequently, the siRNA-expressing cassettes were subcloned into adenoviral vectors. Downregulation of HER2 by adenovirus-mediated RNAi and its effect on SKBR3 cell proliferation were identi- fied again. RESULTS： Two siRNA-expressing plasmids with best interference effect were screened out and HER2 was also efficiently downregulated in SKBR3 cells infected with the adenovirus containing these siRNA-expressing cassettes. Downregulation of HER2 resulted in the increase of cells in G1 phase and the induction of apoptosis. Furthermore, infection of adenovirus inhibited SKBR3 cell growth, which was confirmed by MTT and cell long-term proliferation assays. CONCLUSION： The adenovirus-mediated RNAi could downregulate the HER2 expression efficiently and exert an inhibitory effect on growth of HER2-overexpressingbreast cancer cell.

关 键 词：HER2 RNA干涉 小干涉RNA 腺病毒 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]