HER-2／neu·siRNA表达载体的构建及对人乳腺癌细胞株的影响

目的：探讨siRNA对人乳腺癌细胞株SK—BR-3的HER-2／neu基因表达的影响。方法：以HER-2／neu mRNA序列为模板设计合成2对siRNA序列，构建pGenesil-1-HER-2／neusiRNA重组质粒，转化感受态的大肠杆菌，质粒酶切、测序鉴定。转染SK—BR-3细胞48h后，提取RNA进行RT—PCR，采用方差分析（ANOVA），分析RNA干扰效应。结果：成功构建了pGenesil-1-HER-2／neu siRNA重组质粒，成功转染SK—BR-3细胞。重组质粒抑制HER-2／neu基因的表达接近54．45％（P=0．000）。结论：HER-2／neu siRNA重组质粒明显下调HER-2／neu基因在乳腺癌细胞中的表达。

Vector construction and silencing effects of siRNA to HER-2/neu gene of human breast cancer cell line SK-BR-3

Objective：To investigate the effects of siRNA to HER-2/neu gene of human breast cancer cell line SK-BR-3. Methods： Two pairs of siRNA were designed and synthesized according to the sequence of HER-2/neu mRNA. pGenesil-1-HER-2/neu siRNA recombinant plasmid was then constructed and used to transfected E coll. The constructed plasmids were confirmed by restriction cutting and DNA sequencing. SK-BR-3 cell line was then transfected, and at 48 h later, extracted for total RNA to be detected for expression of HER-2/neu mRNA by RT-PCR. The results was analyzed for gene-silencing effects using ANOVA. Results： pGenesil-l-HER-2/neu siRNA was constructed and SK-BR-3 transfected successfully. RT-PCR analyses found that nearly 54.45% of HER-2/neu mRNA expression in SK-BR-3 was suppressed （ P = 0. 000 ）. Conclusions ： pGenesil-1-HER-2/neu siRNA may significantly inhibit HER-2/neu gene expression in SK-BR-3.