小干扰RNA抑制EB病毒潜伏膜蛋白1表达对鼻咽癌细胞生长的影响  

Effective inhibition of EB virus-encoded latent membrane protein-1 by siRNA in EB virus(+) nasopharyngeal carcinoma cell

作　　者：李刚[1] 李湘平[1] 彭英[2] 刘雄[1] 李晓华[1] 

机构地区：[1]南方医科大学南方医院耳鼻咽喉头颈外科,广州510515 [2]香港大学分子生物学研究所

出　　处：《中华耳鼻咽喉头颈外科杂志》2005年第6期406-410,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家自然科学基金资助项目(30471945);广东省科技计划资助项目(2003C30303)

摘　　要：目的探讨人工诱导RNA干扰技术在EB病毒潜伏膜蛋白1(latentmembraneprotein1,LMP-1)功能研究中的应用价值,观察LMP-1基因沉默对鼻咽癌细胞生长的影响。方法人工合成4条双链不同序列的小干扰RNA(smallinterferingRNA,siRNA),通过脂质体转染导入表达EB病毒基因的鼻咽癌C611细胞后,用半定量逆转录聚合酶链反应(reversetranscriptasepolymerasechainreaction,RTPCR)检测LMP1mRNA水平,筛选出能有效抑制LMP-1表达的siRNA序列。采用噻唑蓝法和流式细胞仪观察LMP1表达抑制后,鼻咽癌细胞增殖、凋亡以及细胞周期的变化。结果导入有效siRNA后,C611细胞中LMP-1mRNA水平下降90%以上,重复转染可使有效干扰时间延长至96h。LMP-1基因抑制后,EB病毒阳性的C611细胞增殖速度最多可下降至32.9%;细胞周期在G0G1期受阻。在同样处理下,EB病毒阴性的HNE2细胞的生长则未受到影响。结论全新的人工诱导RNA干扰技术能够有效抑制LMP1基因的表达,降低鼻咽癌细胞的增殖能力,为鼻咽癌研究和抗肿瘤基因治疗提供了新的思路。Objective To evaluate the application of RNA interference in the study of latent membrane protein-1(LMP-1) in EB virus-positive nasopharyngeal carcinoma (NPC) cell line C611. Observe the effects of LMP-1 silencing on NPC cell growth. Methods Four synthesized small interference RNA (siRNA) were transfected into NPC cell using Oligofectamine^(TM) reagent. LMP-1 mRNA level was determined by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR). Flow cytometry and MTT assay were employed to analyze the effects on cell cycle and proliferation. Results The most effective sequence found out among the 4 candidates. Single dose of this siRNA caused nearly 90% loss of LMP-1 mRNA in C611 cell .The specific inhibition could last for 96 hours if re-transfection was preformed. LMP-1 siRNA treatment resulted in cell cycle arrest at G0-G1 stage in C611, accompany with a reduction of cell proliferation by 32.9%. While EBV-negative NPC cells appeared unaffected. Conclusions These results provided solid testimony that EBV-encoded latent membrane protein-1 was vulnerable to RNA interference and, selective inhibition of LMP-1 had anti-proliferation effect on NPC cell. RNA interference could be a powerful tool in further investigations of LMP-1 and a novel therapeutic strategy for EBV-related NPC patients.

关 键 词：EB病毒潜伏膜蛋白1 癌细胞生长 小干扰RNA 半定量逆转录聚合酶链反应 RNA干扰技术 mRNA水平 LMP-1基因 EB病毒基因 肿瘤基因治疗 人工诱导 细胞周期 脂质体转染 chain RNA序列 流式细胞仪 癌细胞增殖 siRNA 鼻咽癌细胞 

分 类 号：R739.63[医药卫生—肿瘤]