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

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

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Western blotting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—RetroQ—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。

Objective To establish the stable inhibition of HER2/neu expression by vectormediated small hairpin RNA in malignant transformed human bronchial epithelial cell line induced by antibenzo（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 ceils 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-nea 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.