重组腺相关病毒介导的特异性发夹状RNA敲低EB病毒潜伏膜蛋白1表达对鼻咽癌细胞转移的影响  

Influence of suppression of Epstein-Barr Virus-encoded latent membrane protein 1 by rAAV vector mediated RNA interference on metastatic ability of nasopharyngeal cancer cells in vivo

作　　者：刘雄[1] 李刚[1] 张宝[1] 王路[2] 李晓华[3] 李湘平[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]南方医科大学分子生物教研室 [3]解放军第九四医院耳鼻喉科

出　　处：《中华肿瘤杂志》2009年第5期324-329,共6页Chinese Journal of Oncology

基　　金：国家自然科学基金（30471945）;广东省科技计划攻关项目（2003C30303）

摘　　要：目的探讨EB病毒（EBV）潜伏膜蛋白1（LMP-1）对鼻咽癌细胞在体内增殖和转移能力的影响及其可能的机制。方法构建重组腺相关病毒（rAAV）一增强型绿色荧光蛋白（EGFP）和rAAV-shRNA—LMP-1载体。以不同滴度的rAAV—EGFP转染鼻咽癌C666—1细胞，确定最佳转染复数（MOI）。rAAV—shRNA-LMP-1按MOI转染C666—1细胞，采用逆转录聚合酶链反应（RT—PCR）鉴定抑制效率。将体外转染RNA干扰后的C666-1细胞注入裸鼠肝脏包膜下建立鼻咽癌移植瘤模型，观察裸鼠的肝脏成瘤及肝脏、肺脏转移情况。采用免疫组化法检测基质金属蛋白酶9（MMP-9）的表达，分析LMP-1基因沉默对鼻咽癌细胞成瘤和转移能力的影响及机制。结果rAAV—EGFP以5×10。病毒基因组数／细胞转染C666—1细胞，转染效率〉95％。rAAV—shRNA-LMP-1以5×10^4病毒基因组数／细胞转染C666—1后，LMP-1的表达抑制率〉90％。rAAV—shRNA—LMP-1转染组裸鼠的肝脏成瘤体积为（0．2527±0．1152）cm^3，与rAAV—EGFP转染组[（0．2533±0．0754）cm^3]的差异无统计学意义（P〉0．05），但rAAV—shRNA-LMP-1转染组裸鼠的肝转移率（50．0％）和肺转移率（33．3％）明显低于rAAV-EGFP转染组（均P〈0．05）。rAAV—shRNA—LMP-1转染组裸鼠的生存时间为（15．50±2．47）d，明显长于rAAV—EGFP转染组[（11．50±1．22）d，P〈0．05]。免疫组化染色结果显示，LMP-1抑制后，MMP-9的表达明显下调。结论通过rAAV介导RNA干扰序列能有效抑制LMP-1的表达，其可能通过下调MMP-9的表达抑制鼻咽癌细胞的转移，但对鼻咽癌细胞的生长无显著影响。Objective To study the effect of Epstein-Barr virus （EBV） encoded latent membrane protein 1 （ LMP-1 ） on the tumor growth and metastasis of nasopharyngeal carcinoma （NPC） cells in vivo and its possible mechanism. Methods To construct two recombinant adeno-associated virus （rAAV） ： rAAV- shRNA-LMP-1 and rAAV-EGFP （enhanced green fluorescent protein）. Muhiplicity of infection （MOI） was confirmed by using different titre of rAAV-EGFP to transfect a NPC cell line, C666-1. Then C666-1 cells were transfected by rAAV-shRNA-LMP-1 at MOI titre and the inhibiting efficiency of target gene＇s expression was confirmed by reverse transcription polymerase chain reaction （RT-PCR）. C666-1 cells treated by RNAi on LMP-1 in vitro was directly inoculated into the liver via laparotomy under direct vision to establish the animal model of NPC xenograft in liver and lung metastasis from the liver. The biological effect and its mechanism after ＂gene silencing＂ of LMP-1 on NPC cells tumorigenesis and metastasis were observed by the primary intrahepatic tumor formation and lung metastasis and the expression of matrix metalloproteinases-9 （MMP-9） revealed by immunohistochemistry. Results The transfection efficiency was higher than 95% with 5 × 10^4 virus genome （v. g） /cell with rAAV-EGFP. The expression of target gene was inhibited more than 90% , assessed by RT-PCR after transfection with rAAV-shRNA-LMP-1 at a dose of 5 × 10^4 v. g/cell. The primary tumor volume implanted in the liver of rAAV-shRNA-LMP-1 treatment was （0. 2527±0. 1152） cm^3, with no significant difference in comparTison with rAAV-EGFP control group [ （ 0. 2533 ± 0. 0754 ） cm^3, P 〉 0.05 ]. But the rate of intrahepatic tumor formation was 50.0% and the rate of lung metastasis was 33.3% of the rAAV-shRNA-LMP-1 group, significantly lower than those in the rAAV-EGFP group （ P 〈 0.05 ）. The survival time（ 15.50 ± 2.47）d of rAAV-shRNA-LMP-1 group was significantly longer than that in the rAAV-EGFP group [ （

关 键 词：鼻咽肿瘤 重组腺相关病毒 RNA干扰 潜伏膜蛋白1 肿瘤转移 

分 类 号：R686[医药卫生—骨科学]