载体介导的小干扰RNA诱导p53基因的沉默

Vector-Mediated Small Interference RNA Induce Silencing of p53 Gene

目的:筛选有效抑制p53基因表达的靶位点,以研究其在哺乳动物细胞中的功能。方法:用Ambion公司的RNA干扰(RNAi)设计软件设计了3段针对野生型p53基因的长度为19nt的反向重复序列,在人工退火条件下形成双链,将其构建到pSilencer3.1-H1neo载体上,与pcDNA3.1-HA-p53(野生型)载体共转染Cos7及293T细胞,用Westernblot检测HA-p53蛋白质的表达;将pCMV-GFP-p53与p53-RNAi或空载体共转入Cos7细胞,于激光扫描共聚焦显微镜下比较GFP-p53的表达;将p53-RNAi和空载体分别转入BEL-7405-p53-RE-luc+细胞,在不同剂量化疗药物阿霉素的作用下,通过活体成像仪观察内源p53的表达;利用G418(neo)筛选获得稳定整合p53-RNAi的H460细胞(p53野生型)株,用流式细胞仪分析正常及p53基因沉默细胞的周期变化。结果:筛选到一个有效的p53基因沉默位点,并在H460细胞中得到p53基因沉默的稳定细胞株。结论:获得了有效的p53基因RNAi位点,为进一步研究p53基因在细胞中的功能提供了有力的工具。

Objective： To acquire effective target sequence in silencing p53 gene so that detecting the interfering role of p53 gene in mammalian cells. Methods： The three 19 nt small hairpin RNA（shRNA） sequences targeting human wild type p53 gene were chosed using Ambion＇s small interference RNA （siRNA） Target Finder software, the oligonucleotides were annealed and ligated downstream of the H1 promoter. The plasmid of pcDNA3.1-HA-p53 or pCMV-GFP-p53 was trans- fected into the Cos7 or 293T cells with the plasmid of p53-RNAi and control vector respectively. HA-p53 or GFP-p53 protein was detected with monoclonal antibody against HA or by confocal microscope. Then p53-RNAi and control vector were transfected into BEL-7405-p53-RE-luc^＋. After adding doxorubicin to media for i2 hours, the expression of luciferase was detected by Xenogen IVIS 50 system. The p53-RNAi and control vector were also transfected into H460 cells and the stable cell lines were established by selecting with G418. Endogenous p53 mRNA and proteins were detected by Western-blot, cell cycle was analysed by flow cytometer. Results： The effective target sequence in silencing p53 gene was successfully acquired and the expression of p53 was definitely suppressed in the HEK293 72 hours after infection. Con- clusion： We succeeded in finding a seqence targeting p53 gene and silencing its expression, which provided a powerful tool for researching p53 gene function of mammalian cells.