RNA干扰靶向沉默HER2/neu基因对卵巢癌细胞株SK-OV3的影响

RNA干扰靶向沉默HER2/neu基因对卵巢癌细胞株SK-OV3的影响

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

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山东大学学报(医学版)

衣翠华，侯明，李丽珍，张昕，魏军民，黎莉，郝静

山东大学齐鲁医院肿瘤防治研究中心，山东济南 250012

收稿日期:2006-03-29 修回日期:1900-01-01 出版日期:2006-10-24 发布日期:2006-10-24
通讯作者: 衣翠华

YI Cui-hua, HOU Ming, LI Li-zhen,ZHANG Xin,WEI Jun-min,LI Li, HAO Jing

Received:2006-03-29 Revised:1900-01-01 Online:2006-10-24 Published:2006-10-24
Contact: YI Cui-hua

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。

关键词: 基因疗法 , HER2/neu基因, 卵巢癌, RNA干扰

Abstract: 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 transfect 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.

Key words: RNA interference, HER2/neu gene, Ovarian cancer cell, Gene therapy

衣翠华,侯明,李丽珍,张昕,魏军民,黎莉,郝静. RNA干扰靶向沉默HER2/neu基因对卵巢癌细胞株SK-OV3的影响[J]. 山东大学学报(医学版), 2006, 44(10): 1048-1052.

YI Cui-hua,HOU Ming,LI Li-zhen,ZHANG Xin,WEI Jun-min,LI Li,HAO Jing. Effects of RNAi on HER2/neu gene and proliferation of SKOV3 ovarian cancer cells in vitro[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2006, 44(10): 1048-1052.

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