小发夹RNA抑制人上皮生长因子受体-2基因表达细胞株的构建

目的 在经反式二羟环氧苯并芘(BPDE)恶性转化人支气管上皮细胞基础上建立一种载体介导的小发夹RNA(shRNA)抑制人上皮生长因子受体-2(HER2/neu)表达的稳定细胞株.方法 构建靶向干扰HER2/neu shRNA逆转录病毒载体pSIREN-RetroQ-neu,经酶切及测序鉴定后,将重组表达载体经脂质体介导入反式BPDE恶性转化细胞中,同时以阴性片段重组载体转染细胞(阴性对照)和空白细胞(16HBE-T)做对照,经嘌呤霉素筛选阳性转染细胞株.半定量RT-PCR和Westernblotting技术分别检测分析各组细胞中HER2/neu基因mRNA和蛋白表达差异.结果 获得载体介导靶向抑制反式BPDE恶性转化细胞中HER2/neu基因表达的细胞株.pSIREN-RelroQ-neu阳性转染细胞组分别比较阴性对照和空白细胞组HER2/neu mRNA表达量均下降(平均灰度值分别为0.114±0.003、0.186±0.001、0.182±0.015),其差异有统计学意义(t值分别为39.154、7.564,P值均<0.05),而阴性与空白对照组间相比差异无统计学意义(t=-0.409,P>0.05).pSIREN-RetroQ-neu转染细胞组相比阴性对照和空白细胞组HER2/neu蛋白表达明显下降,其抑制率分别达40%和39%.结论 成功构建pSIREN-RetroQ-neu重组质粒,并能有效抑制反式BPDE恶性转化细胞中HER2/neu表达.

Construction of cell line of small hairpin RNA-mediated inhibition of HER2/neu gene expression

Objective To establish the stable inhibition of HER2/neu expression by vector-mediated small hairpin RNA in malignant transformed human bronchial epithelial cell line induced by anti-benzo( a ) pyrene-trans-7,8-dihydrodiol-9,10-epoxide ( anti-BPDE ). Methods The pSIREN-RetroQ-neu recombinant vector targeting HER2/neu was constructed and confirmed by restriction and sequencing analysis,then it was transfected into anti-BPDE malignant transformed 16HBE cells (16HBE-T) through lipofectamine 2000. The control groups included the 16HBE-T cells transfected with negative control vector (negative control) and 16HBE-T. The cells transfected with vectors were screened by puromycin. The HER2/ neu mRNA and protein expressions in the vector-transfected 16HBE-T cells were detected by RT-PCR and Western blot method respectively. Results The pSIREN-RetroQ-neu recombinant vector which inhibited HER2/neu mRNA and protein expressions in the 16HBE-T was constructed. The level of HER2/neu mRNA in the 16HBE-T cells transfected with pSIREN-RetroQ-neu was significantly reduced as compared to the negative control and blank control cells (0.114 ± 0.003 vs. Blank control 0.186 ± 0. 001, t = 39.154, P < 0. 05;and negative control 0.182 ± 0.015 ,t = 7.564, P < 0.05 ), while its level did not differ significantly between negative control cells and blank control of 16HBE-T (t = -0.409 ,P >0.05 ). HER2/neu protein level in pSIREN-RetroQ-neu transfected cells was inhibited by 40% and 39% respectively. Conclusion Plasmid-based shRNA expression systems targeted against HER2/neu gene were generated successfully, which resulted in down-regulation of HER2/neu gene expression in the 16HBE-T efficiently.