shRNA沉默NRP-1基因抑制鼻咽癌CNE-2Z细胞生长的体内外研究  

NRP-1/shRNA silencing suppresses nasopharyngeal carcinoma cell growth in vitro and in vivo

作　　者：孙瑾[1] 王亮[1] 娄卫华[1] 曹华[1] 田秀芬[1] 桑建中[1] 

机构地区：[1]郑州大学第一附属医院咽喉头颈外科,450052

出　　处：《中华耳鼻咽喉头颈外科杂志》2015年第4期312-317,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

摘　　要：目的 构建靶向神经纤毛蛋白质1（Neuropilin-1,NRP-1）的短发卡RNA （shRNA）表达载体,观察NRP-1对人鼻咽癌细胞系CNE-2Z细胞生长增殖的影响.通过体内裸鼠成瘤实验观察最优质粒对肿瘤的抑制效果.方法 根据NRP-1 mRNA序列设计并构建3个靶向NPR-1基因,并含荧光素标记的shRNA真核表达载体（pSilencer-shRNA1、pSilencer-shRNA2和pSilencer-shRNA3）;以脂质体Lipofectamine 2000转染的方法将shRNA表达质粒转导入CNE-2Z细胞及裸鼠瘤体内,于激光共聚焦荧光显微镜观察shRNA转染表达情况;以四甲基偶氮唑盐（MTT）法检测细胞增殖活性;实时定量PCR（RT-PCR）检测NRP-1基因在mRNA水平表达的抑制作用;Western印迹法在蛋白水平上检测对目的基因的封闭效果.体内裸鼠成瘤实验观察质粒对肿瘤的抑制效果.结果 质粒成功转染CNE-2Z细胞及瘤体,可见大量的癌细胞表达绿色荧光.MTT实验证明NRP-1 shRNA抑制CNE-2Z细胞的生长增殖;RT-PCR结果表明NP-1的mRNA表达降低;Western印迹法结果验证目的基因蛋白同时表达下调;体内裸鼠成瘤实验证实转染6周后,转染组瘤体积生长较阴性对照组、空白对照组明显受到抑制[（0.599 ±0.002）比（1.141 ±0.013）、（1.165 ±0.308）cm^3,均P＜0.05],抑瘤率为48.6％.结论 表达shRNA的NRP-1基因特异的真核表达质粒能有效转染体内、外鼻咽癌细胞,沉默NRP-1基因能有效抑制鼻咽癌细胞生长增殖.为鼻咽癌基因治疗奠定了良好的实验基础。Objective To investigate the effect of RNA interference （RNAi） targeting NRP-1 gene on the growth and proliferation of nasopharyngeal carcinoma（NPC） CNE-2Z cells.Methods Short hairpin RNA（shRNA） plasmids targeting NRP-1 were designed and synthesized.These plasmids were respectively transfected into human NPC CNE-2Z cells and xenograft tumors in nude mice.The expressions of Fluorescein-labeled plasmids in NPC CNE-2Z cells and xenograft tumors were observed by fluorescence microscopy.Cell proliferation was detected by MTT assay.The inhibitory effects on target genes were evaluated with RT-PCR and Western blotting,respectively.The inhibitory effect of plasmids with the most effective sequence on xenograft tumors in nude mice was observed.Results CNE-2Z cell proliferation was significantly inhibited by NRP-1/shRNA silencing.RT-PCR showed NRP-1 mRNA expression was significantly decreased.Western blotting demonstrated the NRP-1/shRNA silencing can effectively inhibit the expression of target proteins in CNE-2Z cells.After six weeks,there were significant differences in the mean tumor volumes in nude mice between plasmid group and negative control group [（0.599 ± 0.002） vs （1.141 ± 0.013） cm^3,P 〈 0.05] or blank control group [（0.599 ± 0.002） vs （1.165 ± 0.308） cm^3,P 〈0.05],and the inhibitory rate of tumor growth was 48.6%.Conclusion RNA interference targeting NRP-1 can remarkably inhibit the growth of CNE-2Z cells in vitro and in vivo.

关 键 词：神经纤毛蛋白质1 鼻咽肿瘤 基因治疗 裸鼠成瘤 NEUROPILIN-1 

分 类 号：R739.63[医药卫生—肿瘤]