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目的研究应用载体介导的RNA干涉技术特异性干扰Raf-1基因在鼻咽癌细胞株HNE1中的表达以及对其生物学行为的影响。方法构建利用U6启动子转录功能的shRNA的载体,在U6启动子下游分别插入含针对Raf-1基因不同位点的特异性RNA干扰序列-67bp寡核苷酸片段,并编号命名为:pshuttle-Raf-1-a(225),pshattle-Raf-1-b(358)和pshuttle-Raf-1-c(474)。将构建的质粒通过脂质体转染人鼻咽癌细胞株HNE1,用RT-PCR分析Raf-1基因的mRNA的表达,流式细胞仪检测转染后HNE1细胞的凋亡率。结果3种含有针对Raf-1基因不同特异性序列的质粒转染后均能干扰该基因在HNE1细胞中的表达,其中以pSIRENshuttle-Raf-1-b(358)作用最为明显。RT-PCR检测Raf-1基因mRNA表达量下降,流式细胞仪检测细胞凋亡率增加,达到65%。结论应用载体介导的RNAi技术可特异性干扰Raf-1基因在鼻咽癌细胞HNE1中的表达,使该细胞凋亡率增加,Raf-1基因相对应的mRNA表达下降。
Objective To investigate the effect of vector-mediated RNA interference on the expression of Raf-1 gene in nasopharyngeal carcinoma cell line HNE1 and its biological behavior. Methods The shRNA vector using the U6 promoter transcription function was constructed. The downstream of U6 promoter was inserted into the specific RNA interference sequence -67bp oligonucleotide fragment containing different sites of Raf-1 gene and named as pshuttle- Raf-1-a (225), pshattle-Raf-1-b (358) and pshuttle-Raf-1-c (474). The constructed plasmid was transfected into human nasopharyngeal carcinoma cell line HNE1 by lipofectamine. The mRNA expression of Raf-1 gene was analyzed by RT-PCR. The apoptosis rate of HNE1 cells was detected by flow cytometry. Results All three plasmids containing different specific sequences of Raf-1 gene could interfere with the expression of this gene in HNE1 cells. Among them, pSIRENshuttle-Raf-1-b (358) was most effective. The mRNA expression of Raf-1 was down-regulated by RT-PCR, and the apoptosis rate increased by 65% with flow cytometry. Conclusion The vector-mediated RNAi technique can specifically interfere with the expression of Raf-1 gene in the nasopharyngeal carcinoma cell line HNE1, leading to an increase in the apoptosis rate and a corresponding decrease in the mRNA expression of Raf-1 gene.