siRNA干扰对SKBr3细胞株HER2表达的影响

Effect of small interference RNA on expression of the epidermal growth factor receptor 2 in SKBr3 cell line

目的探讨siRNA干扰对人乳腺癌细胞株SKBr3表皮生长因子受体2(HER2)表达的影响。方法构建2个针对HER2基因表达短发夹状siRNA的真核表达载体(psiRNA-HER2),并转染人乳腺癌细胞株SKBr3,采用RT-PCR法和流式细胞仪检测转染后48h HER2 mRNA和蛋白表达,从中筛选出基因沉默效果好的靶位点。同时构建针对绿色荧光蛋白(GFP)基因的短发夹状siRNA的真核表达载体psiRNA-GFP作为阳性对照。结果psiRNA-HER2-1、psiRNA-HER2-2转染后均能抑制人乳腺癌细胞株SKBr3 HER2 mRNA和蛋白表达,抑制率分别为32.0%和65.5%。荧光显微镜下见psiRNA-GFP转染后明显抑制GFP的表达。结论HER2 siRNA真核表达载体可在人乳腺癌细胞株SKBr3中产生RNA干扰效应,不同的siRNA下调mRNA和蛋白表达的效率不同。作为基因沉默的一个重要工具,siRNA干扰有望成为治疗乳腺癌的一种新方法。

Objective To study the inhibition of epithelial grow factor receptor 2 （HER2） by small interference RNA （siRNA） on the effect of HER2 in SKBr3 cell line of human breast cancer. Methods Eukaryotic expression vector targetingH-ER2gene, namedpsiRNA-HER2-1/psiRNA-HER2-2, was constructed and transfected into cultured human breast carcinoma cell line SKBr3 via lipofeetamine2000; The inhibited effects on HER2 gene were determined by semi-quantitative reverse transcription PCR and flow eytometry. At the same time, the eukaryotic siRNA expression vector targeting GFP gene, named psiRNA-GFP was constructed as the positive control. Results Both psiRNA-HER2-1 and psiRNA-HER2-2 can inhibited HER2 gene expressing. The inhibit rate of psiRNA-HER-1 is 32.0% and the rate of psiRNA-HER-2 is 65.5% ; psiRNA-GFP strongly decreased the green fluorine protein expression under fluoroscope. Conclusion The eukaryotic siRNA expression vector targeting HER2 can down-regulate the expression of HER2 mRNA and protein in SKBr3 cell line and the clown-regulate rate varies based on different siRNA. As an important tool of gene silencing, siRNA is likely to be a new method in the treatment of human breast cancer.