siRNA抑制HER2/neu过表达肺腺癌细胞生长和增殖

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Western Blot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。

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

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 mRAN and protein were detected by RT-PCR and Western Blot.FCM analysis,MTT method and colony forming test were applied to measure cell 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 S1 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 cel1.Cells transected with the plasmids expressing siRNA targeting HER2/neu gene increased accumulation of cells in G_0/G_1 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 G_0/G_1 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.