利用高效siRNA表达载体抑制结肠癌细胞Notch1基因表达的研究

Suppression of Notchl gene expression in colon cancer cells by use of high - efficient siRNA expression vector

目的 探讨利用靶向Notch1基因的高效siRNA真核表达载体抑制结肠癌细胞中Notch1表达的可行性.方法 根据Notch1 cDNA编码序列,设计并合成靶向Notch1基因的4对特异性RNA干扰片断,将其克隆入pSilencer4.1-CMV neo载体中.将重组载体及其阴性对照载体转染结肠癌细胞SW480,采用逆转录-多聚酶链反应（reverse transcription-polymerase chain reaction,RT-PCR）检测各组细胞中Notch1 mRNA表达水平,筛选获得能够高效抑制结肠癌细胞Notch1表达的siRNA真核表达载体.结果 成功构建了4个靶向Notch1基因的siRNA真核表达载体;转染结肠癌细胞SW480后,与对照组相比各干扰组细胞中Notch1 mRNA表达水平均明显下降,其中尤以psiNotch1-A组下降最为明显.结论 成功筛选出能够在结肠癌细胞中高效抑制Notch1基因表达的特异性siRNA真核表达载体,为进一步利用siRNA靶向Notch1治疗结肠癌奠定了基础.

Objective To explore the feasibility of inhibiting Notchl expression in colon cancer cells by use of siRNA eukaryotic expression vector targeting Notchl gene. Methods According to Notchl cDNA sequence ,4 specific RNA interference （RNAi） fragments targeting Notchl gene were de- signed and synthesized ,which were then cloned into pSilencer4.1 -CMV neo vector. Colon cancer SW480 cells were transfected with the recombinant plasmids and negative control respectively. The Notchl mRNA level in each transfected group was determined by reverse transcription - polymerase chain reaction （ RT - PCR） to select the efficient siRNA expression vector that could greatly suppress the Notchl expression in colon cancer cells. Results Four specific siRNA eukaryotic expression vectors targeting Notchl gene were constructed successfully. The level of Notchl mRNA in the cells transfected with siRNA vectors, especially in the cells transfected with psiNotchl -A, was much deceased as compared with that in the control cells. Conclusion Specific siRNA eukaryotic expression vector that could effectively suppress the expression of Notchl gene in colon cancer cells was obtained,which provides a basis for the treatment of colon cancer by using Notchl - targeted siRNA.