RNA干涉鼻咽癌细胞Raf-1基因表达的实验研究  

Specific inhibition of Raf-1 gene expression in HNE1 by RNA interference

作　　者：刘立中[1] 梁传余[1] 窦艳玲[1] 田聆[2] 魏于全[2] 文艳君[2] 李炯[2] 邓红新[2] 阚兵[2] 

机构地区：[1]四川大学华西医院耳鼻咽喉科 [2]四川大学肿瘤生物治疗国家重点实验室

出　　处：《中华耳鼻咽喉头颈外科杂志》2006年第11期805-808,共4页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：973基因治疗的应用基础研究(2004CB518800)

摘　　要：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 silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference （RNAi） technique. Methods The vector containing the human U6 promoter was used for targeted gene silencing when a dsDNA oligonucleotide encoding an appropriate shRNA was ligated into the vector, and 67nt oligonucleotide fragment was inserted into the downstream of the U6 promoter. Plasmids containing different Raf-1 target sequences [ （1）pshuttle-Raf-1-a （ 225 ）, （2）pshattle-Raf-1-b （ 358 ） and （3） pshuttle-Raf-1-c（474） ] ,were transfected into HNE1 cells. Expression of Raf-1 mRNA was assayed by RT-PCR. Apoptosis were determined by cytometry. Results Vector-based RNAi had advantages over antisense RNA because it could be delivered to the target cell more efficiently, and effect could last longer. Raf-1 expression could be inhibited by plasmid-expressed shRNA. Three different targeting sequences were selected from Raf-1 gene, and the inhibitory effect of pSIREN shuttle-Raf-1-b （358） was biggest. Condusion Raf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.

关 键 词：鼻咽肿瘤 原癌基因蛋白质 c-caf类 基因 

分 类 号：R739.63[医药卫生—肿瘤]