针对HER2/neu的siRNA对非小细胞肺癌细胞生长的抑制作用

Inhibition of tumor growth of non-small cell lung cancer by small interfering RNA targeting to HER2/neu gene

目的:构建针对HER2/neu的RNA干涉载体,观察对HER2/neu表达抑制及抑制后对非小细胞肺癌生长的影响.方法:设计和合成长度为64nt的脱氧寡核苷酸链,退火互补成双链,克隆至pSUPER质粒载体,转化DH5α菌株,提取质粒,转染过表达HER2/neu的肺腺癌细胞系SPCA1,RTPCR检测HER2/neu的mRNA表达,Westernblot和间接免疫荧光法检测HER2/neu的蛋白表达,FCM分析细胞周期变化,MTT法观察细胞生长.结果:肺腺癌细胞系SPCA1存在HER2/neu过表达;成功构建了HER2/neu特异性RNA干涉载体pSUPERsiHER2;瞬时转染SPCA1细胞,HER2/neu的mRNA及蛋白表达均降低;G1期细胞较亲代增加9.9%;肿瘤细胞增殖速度减慢(P<0.05).结论:成功构建了HER2/neuRNA干涉载体,转染后可有效降低肺腺癌细胞系SPCA1HER2/neu的mRNA转录及蛋白水平表达,阻滞转染细胞于G1期,造成转染细胞生长速度减慢,这有望为肺癌基因治疗提供一种选择手段.

AIM： To construct RNA interference （RNAi） vector to down-regulate HER2/neu gene and study the RNAi effect on the cell cycle and tumor growth of non-small cell lung cancer. METHODS： Oligonucleotides of 64 base pairs for hairpin RNA targeting HER2/neu were designed, chemically synthesized and annealed, and cloned into pSUPER vector. After identified by restriction digestion, the right vectors were transiently transfected into SPC-A-1 cells, a human lung adenocarcinoma cell line. The HER2 mRNA was detected by RT-PCR, and the protein detected by Western blot and indirect immunolluorescence staining. FCM analysis and MTT method were applied to measure cell cycle and cell growth respectively. RESULTS： SPC-A-1 cells had an overexpression of HER2. The vector of RNAi which can interfere HER2/neu gene was successfully constructed, and compared with parent cells, the vector can increase the number of cells in G0/GI phase by 9.9%, and inhibit the tumor cell growth （ P 〈 0.05 ）. CONCLUSION： We successfully constructed an expressing hairpin RNA against HER2/neu vector. The vector can effectively silence HER2/neu gene, increase the number of cells in G0/GI phase, and then slow down the growth of tumor cells. This may be a useful therapeutic strategy for NSCLC over-expressing HER2/ neu.