应用可调控Notch1干扰载体诱导HeLa细胞增殖抑制  

Effect of CRE-Dependent RNA Interference Targeting Notch1 on Proliferation of Cervical Cancer Cell Line HeLa

作　　者：俞海[1] 黄盛林[1] 赵小平[1] 卢健[1] 钱关祥[1] 葛盛芳[1] 

机构地区：[1]上海交通大学医学院生物化学与分子生物学研究室人类基因治疗研究中心,上海200025

出　　处：《癌症》2007年第2期148-153,共6页Chinese Journal of Cancer

基　　金：国家重大基础研究项目(No.2004CB518804)~~

摘　　要：背景与目的:Notch信号转导途径与细胞增殖与分化的调控密切相关,它的功能失调在肿瘤细胞的增殖分化中发挥着重要的作用。本研究构建了针对Notch1的可调控RNA干扰载体,并从分子水平及细胞水平上研究其对HeLa细胞的影响。方法:利用受CRE重组调控的RNA干扰载体pSico和pSicoR构建针对GAPDH和Notch1的shRNA表达载体,以pBS185-CRE作为CRE蛋白的表达载体,将HeLa细胞分为pSico、pSico/CRE、pSicoR和pSicoR/CRE4组,分别转染相应质粒,RT-PCR和Westernblot法检测干扰效率,CBF-1荧光素酶报告载体检测细胞内活性Notch信号水平,MTS实验检测细胞增殖状态。结果:各组细胞在转染重组质粒pSico(R)-GAPDH和pSico(R)-Notch1后,EGFP的表达均表现出明显的CRE依赖性;RT-PCR和Westernblot实验显示,pSico(R)-Notch1载体能在CRE蛋白的调控下抑制Notch1基因的表达,CBF-1荧光素酶报告载体实验可见,Notch1的干扰作用使细胞内Notch信号水平下降;MTS实验可见Notch1干扰引起的HeLa细胞增殖抑制。结论:由可调控RNA干扰载体介导的CRE依赖性Notch1表达降低能降低细胞内Notch信号水平并抑制HeLa细胞增殖。BACKGROUND ＆ OBJECTIVE： Notch signal transduction pathway mediates cell differentiation and proliferation. Its dysfunction is supposed to be involved in tumorigenesis and development. This study was to construct a course recombination enzyme （CRE）-dependent short hairpin RNA （shRNA） expression plasmid targeting Notch1, and investigate its effect on proliferation of cervical cancer cell line HeLa. METHODS： RNA interfering vectors pSico and pSicoR were used to construct CRE-dependent shRNA expression plasmids targeting GAPDH and Notch1 ; pBS185-CRE was used as an expression vector of CRE. HeLa cells were divided into 4 groups and transfected with pSico, pSico/CRE, pSicoR, and pSicoR/CRE, respectively. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blot were carried out to assess the efficiency of RNA interference （RNAi）, and intracellular Notch signal level was tested by CBF-1 reporter plasmid. The proliferation of HeLa cells after CRE-dependent Notch1 RNAi was detected by MTS assay. RESULTS, After transfection of pSico （R）-GAPDH and pSico （R）-Notchl, CRE-dependent green fluorescent cells were detected ; Notch1 expression was inhibited; intracellular Notch1 signal level was decreased; the proliferation of HeLa cells was suppressed. CONCLUSION： RNAi of Notch1 mediated by CRE-dependent shRNA expression plasmid can down-regulate intracellular Notch1 signal level and suppress the proliferation of HeLa cells.

关 键 词：NOTCH RNA干扰 HELA细胞 CRE 

分 类 号：Q78[生物学—分子生物学]