hTERT双链RNA对肺癌细胞端粒酶的抑制作用  

Inhibitory Effect of hTERT dsRNA on Telomerase Activity in Lung Carcinoma Cell Line A549

作　　者：田凤军[1] 王智勇[2] 马俊义[3] 赵云霞[1] 卢炜[1] 

机构地区：[1]河北医科大学第三医院呼吸内科,河北石家庄050051 [2]河北医科大学第三医院危重医学科,河北石家庄050051 [3]河北医科大学第二医院呼吸内科,河北石家庄050051

出　　处：《癌症》2005年第3期257-261,共5页Chinese Journal of Cancer

基　　金：河北省卫生厅青年基金资助项目(No.03082)~~

摘　　要：背景与目的:RNA干涉(RNAinterference,RNAi)是基因功能研究新技术,其机制是通过双链RNA(double-strandedRNA,dsRNA)与靶RNA结合并将其降解。本实验探讨dsRNA对肺癌细胞端粒酶逆转录酶组分(humantelomerasereversetranscriptase,hTERT)抑制的可能性和特异性,了解其对肺癌细胞增殖的影响,观察其是否对真核细胞有非特异杀伤作用,探讨其在肿瘤研究和治疗方面应用的可能性。方法:RT-PCR和PCR扩增hTERT基因两个外显子和一个内含子部分序列,采用正、反义cDNA链串联形式构建dsRNA真核表达载体,转染肺癌A549细胞。RT-PCR检测hTERTmRNA表达,Westernblot检测hTERT蛋白表达,TRAP法检测端粒酶活性,倒置显微镜观察细胞形态变化,绘制细胞生长曲线了解细胞增殖情况。结果:hTERT外显子的两段dsRNA作用肺癌A549细胞后,都表现出hTERT基因表达抑制、端粒酶活性下降、细胞增殖受到抑制,而且,dsRNA未引起细胞非特异死亡。结论:hTERTdsRNA能特异使hTERT基因沉默,对肺癌细胞A549端粒酶活性和细胞生长具有明显的抑制作用,有可能成为肺癌基因治疗的新方法。BACKGROUND & OBJECTIVE: RNA interference (RNAi) is a new technology in gene study. The mechanism of RNAi is that double-stranded RNA (dsRNA) can band target mRNA and decompose it. This study was to assess possibility and specificity of dsRNA on suppressing human telomerase reverse transcriptase (hTERT) in lung carcinoma cells, investigate its effect on cell proliferation to confirm whether it has unspecific killing activity on mammalian cells, and explore its application in lung cancer research and treatment. METHODS: Sequences of 2 exons and 1 intron of hTERT gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR) or PCR. The sense and antisense cDNA sequences were connected in a tandem manner, and the whole fragment was inserted into pCI-neo mammalian expression vector to construct the dsRNA expression vector, and then transfected into lung carcinoma cell line A549. The expression of hTERT was detected by RT-PCR and Western blot. Telomerase activity was measured by telomerase repeat amplification protocol (TRAP). Cell morphology was observed, and cell proliferation was assessed under invert microscope. RESULTS: After transfection of 2 exon fragments of hTERT dsRNA, mRNA and protein expression of hTERT and telomerase activity in A549 cells were suppressed, cell proliferation was markedly inhibited. Meanwhile, dsRNA didn't show unspecific toxic activity on A549 cells. CONCLUSIONS: hTERT dsRNA can specifically silent hTERT gene, inhibit telomerase activity and proliferation of A549 cells. hTERT dsRNA might be a potential method of gene therapy for lung cancer.

关 键 词：双链RNA RNA干涉 人端粒酶逆转录酶 肺肿瘤 

分 类 号：R734.2[医药卫生—肿瘤]