siRNA抑制HER2/neu过表达肺腺癌细胞生长和增殖  

Inhibition of cell proliferation and colony formation in overexpression HER2/neu adenocarcinoma cell of lung by vector based-small interfering RNA

作　　者：任新玲[1] 钱桂生[1] 付海京[2] 鲍炜[3] 王涛[2] 张瑞[2] 张立红[2] 贾林涛[2] 杨安钢[3] 

机构地区：[1]第三军医大学新桥医院全军呼吸内科研究所,重庆400037 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032 [3]第四军医大学基础部免疫学教研室,陕西西安710032

出　　处：《中国癌症杂志》2006年第5期333-336,340,共5页China Oncology

基　　金：国家重大基础研究计划项目"973"(2004CB518805);教育部"长江学者和创新团队发展计划"项目(IRT0459);国家自然科学基金(30500592)

摘　　要：背景与目的:30%NSCLC存在HER2/neu过表达,与肿瘤发生、转移、肿瘤血管形成、抗凋亡及化疗耐药等有关。本文通过RNA干涉抑制肺腺癌细胞SPC-A-1 HER2/neu过表达,观察肿瘤细胞周期、增殖及集落形成能力的变化。方法:构建针对HER2/neu的siRNA重组质粒,稳定转染SPC-A-1,通过RT-PCR和W esternB lot检测HER2/neu表达;FCM分析细胞周期;MTT法观察细胞增殖,绘制细胞生长曲线;平板集落形成实验检测肿瘤细胞的集落形成能力。结果:成功构建了HER2/neu的siRNA重组质粒,稳定转染靶细胞后可显著降低HER2/neu表达;与亲代细胞相比,G0/G1期细胞增加17.1%,S期细胞减少12.8%;细胞生长速度减慢,集落S1抑制率达到49.0%。结论:针对HER2/neu的siRNA可以显著降低肺腺癌细胞过表达HER2/neu,肿瘤细胞阻滞于G0/G1期,造成细胞增殖减慢,集落形成能力明显下降。因此,siRNA对过表达HER2/neu肺癌的治疗具有潜在应用价值。Background and purpose： The HER2/neu oncogene is overexpressed in 30% of non-small-cell lung cancer （NSCLC） , especially in adenocarcinoma cell of lung , and may be related to carcinogenesis, metastasis, anti-apoptosis and chemotherapy resistance. Therapeutic agents against HER2/neu have been intensively investigated over the past decade. Here we construct RNA interference recombiment plasmid to down-regulate HER2/neu gene and study the effect of siRNA of HER2/neu on the cell cycle, tumor growth , and colony forming capability of SPC-A-1 adenocarcinoma cell line. Methods： The plasmids expressing siRNA against HER2/neu were constructed and stably transected into HER2/neu-overexpressing SPC-A-1 cell with Lipofectamine^TM 2000 and positive single colonies were screened out with G418. The HER2/ neu reRAN and protein were detected by RT-PCR and Western Blot. FCM analysis, MTF method and colony forming test were applied to measure ceil cycle, cell growth and the ability of cancer cell to form colonies. Results： The plasmids expressing siRNA against HER2/neu were successfully constructed, and one of the colonies named SI was found to express siRNA against HER2/neu effectively because the expression of HER2/neu in this colony was significantly decreased compared with that of parent cell. Cells transected with the plasmids expressing siRNA targeting HER2/neu gene increased accumulation of cells in G0/G1 phase by 17.1% with a decrease in the percentage of cells in S-phase by 12.8%, and exhibited slower proliferation, and inhibited of colonies by 49.0%, compared with those of the partent cells. Conclusions： We successfully constructed recombineut plasmid siHER-2 against HER2/neu. siRNA exhibit slower proliferation, increased G0/G1 arrest, and decreased tumor growth and colony formation. The data indicated that siRNA-mediated gene inhibition of HER2/neu may be a useful therapeutic strategy for HER2/neu-overexpression in NSCLC.

关 键 词：SIRNA HER2/NEU 肺腺癌 细胞增殖 集落形成 细胞周期 

分 类 号：R734.2[医药卫生—肿瘤]