RNA干扰靶向沉默HER2/neu基因对卵巢癌细胞株SK-OV-3的影响  

Effects of RNAi on HER2/neu gene and proliferation of SK-OV-3 ovarian cancer cells in vitro

作　　者：衣翠华[1] 侯明[1] 李丽珍[1] 张昕[1] 魏军民[1] 黎莉[1] 郝静[1] 

机构地区：[1]山东大学齐鲁医院肿瘤防治研究中心,山东济南250012

出　　处：《山东大学学报（医学版）》2006年第10期1048-1052,共5页Journal of Shandong University:Health Sciences

摘　　要：目的:观察siRNA表达质粒稳定转染靶向沉默HER2/neu基因对SK-OV-3卵巢癌细胞株的影响。方法:针对HER2/neu基因序列构建短发夹状siRNA真核表达载体(pGenesil-1-HER2/neu),转染卵巢癌细胞株SK-OV-3,G418筛选出稳定转染株后,采用RT-PCR和Western Blot法观察HER2/neu基因的沉默效果;CCK-8比色分析检测细胞体外增殖活力;流式细胞仪检测细胞周期。结果:测序证实成功构建HER2/neu的2个短发夹状siRNA真核表达质粒;稳定转染SK-OV-3细胞后,转染了特异性HER-2/neu siRNA的细胞HER-2/neu mRNA及p185蛋白水平均明显低于亲本细胞及转染无义对照序列的细胞;CCK-8比色分析显示HER-2/neu基因沉默的细胞存活分数明显低于HER-2/neu基因高表达的细胞(P=0.000);流式细胞仪细胞周期分析也显示,HER-2/neu基因沉默的细胞处于凋亡状态及非增殖期的比例明显增加(P均<0.01),而处于合成期的比例却显著减少(P≤0.001)。结论:靶向HER2/neu基因的短发夹状siRNA可从转录及翻译水平有效地沉默HER-2/neu基因;HER-2/neu基因沉默的SK-OV-3卵巢癌细胞增殖明显受抑制。Objective： To investigate the effects of HER2/neu gene silencing on SK-OV-3 ovarian cancer cells by RNA interference. Methods： Design and synthesize two siRNAs based on the sequence of HER2/neu mRNA. They were separately subcloned into the plasmid of pGenesil- 1 containing U6 promoter. The pGenesil- 1 vectors of the RNA interference eukaryotic expression specific to HER2/neu gene were constructed. The pGenesil-1 vectors were transfected into SK-OV-3 ovarian cancer cells with METAFECTENE in vitro. The positive cell clones were obtained after being screened with 250 μg/ml G418. Then the effects of RNAi on the expression of HER2/neu gene were detected by RT-PCR and Western Blot. The cellular growth activities were assayed by CCK-8 colorimetry and flow cytometry in vitro. Results： DNA sequencing of the plasmids verified the successful construction of the HER2/neu siRNA vectors. Compared with the blank and the control group, the expressions of HER2/neu mRNA and protein 185 were remarkably down-regulated in the RNAi groups; and the cell proliferations decreased in the RNAi groups. Conclusion： We successfully construct the recombinant plasmids of siRNAs specific to HER2/neu gene and tmnsfect them into the SK-OV-3 ovarian cancer cells in vitro. The RNAi inhibits the expression of HER2/neu mRNA and protein 185, and subsequently suppresses the proliferation of SK-OV-3 cells.

关 键 词：RNA干扰 HER2/NEU基因 卵巢癌 基因疗法 

分 类 号：R730.54[医药卫生—肿瘤]