EZH2对鼻咽癌细胞增殖和侵袭影响的研究  

Effects of enhancer of zeste homolog （EZH2） downregnlation on the proliferation and invasion of nasopharyngeal carcinoma cell and the possible mechanisms

作　　者：梁碧君[1] 李湘平[1] 鲁娟[1] 林少雄[1] 刘雄[1] 李刚[1] 张宝[2] 王路[1] 罗花南[1] 万仁强[1] 田文栋[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]南方医科大学基因工程研究所

出　　处：《中华耳鼻咽喉头颈外科杂志》2012年第4期298-304,共7页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：2011年国家自然科学基金（81172053）;2009年广东省自然科学基金（9151051501000093）;2010年广东省自然科学基金（10151051501000092）

摘　　要：目的探讨果蝇zeste基因增强子人类同源物（enhancer of zeste homolog2，EZH2）对鼻咽癌细胞增殖及侵袭能力的影响及其分子机制。方法构建特异性的短发夹状（short hairpin RNA，shRNA）EZH2慢病毒干扰载体，转染293FT细胞包装成慢病毒后，感染鼻咽癌细胞株5—8F。采用四甲基偶氮唑蓝法、平板克隆形成实验和流式细胞术检测细胞增殖状态及细胞周期的改变，利用细胞划痕实验及Invasion Chamber侵袭小室测定法检测迁移和侵袭能力变化，荧光定量PCR及蛋白免疫印迹法检测转染EZH2基因后的mRNA和蛋白表达水平改变及上皮间质转化相关标志物的变化。结果成功构建EZH2 shRNA慢病毒干扰载体，包装的慢病毒感染鼻咽癌细胞株5—8F后，EZH2的mRNA和蛋白表达水平显著下凋。四甲基偶氮唑蓝法显示，细胞接种96h后，5-8F／shEZH2组的生长速度较空载组显著下降（P〈0．001），平均（x^-±s，下同）细胞克隆形成率为（31．56±7．49）％明显小于空载组的（84．44±7．12）％，差异有统计学意义（P=0．001）；细胞周期结果表明，沉默EZH2表达的细胞增殖受抑，主要通过诱导G0／G1期阻滞，减少s期细胞的比例；基因转染沉默EZH2后，鼻咽癌细胞的运动迁移能力显著下降，5-8F／shEZH2组细胞划痕48h的相对迁移率为（0．58±0．05）明显低于空载组的（0．81±0．02），差异有统计学意义（P〈0．000）；体外侵袭能力明显降低，5-8F／shEZH2组穿膜细胞数（72．23±4．08）个，与其相对应空载组（150．95±16．27）个比较，明显减少（P〈0．000）；5-8F／shEZH2细胞的上皮标志物上皮性钙黏附蛋白（E—cadherin）、细胞角蛋白18（Keratin18）的mRNA水平分别上调1．77倍、1．58倍，间质标志物β-连环蛋白（β—catenin）、神经性钙黏附蛋白（N-cadherin）分别下调18．04％、41．18％；蛋白免疫印迹证实此四者的蛋白水平Objective To investigate the effects of the enhancer of zeste homolog 2 （EZH2） gene on cell growth and invasion of the nasopharyngeal carcinoma （NPC）. Methods Recombinant lentivirus vector for shRNA delivery of EZH2 was constructed and transfected into 293FT cells. After collecting the viral panicles, the NPC cell line 5-8F cells were transfected. The effects of EZH2 silence on cell proliferation and cell cycle were detected using MTF assay, plate colony formation assay and flow cytometry. The migration and invasion of 5-8F cells were determined by wound healing assay and matrige] invasion assay, respectively. The expressions of EZH2 and epithelial-mesenchymal transition （EMT）-related markers at mRNA and protein levels were examined by real-time PCR and Western blot respectively. Results The expressions of EZH2 mRNA and protein in the transfected 5-8F cells were obviously reduced. MTT assay showed that EZH2 downregulation significantly inhibited the growth of 5-8F/shEZH2 cells （ P 〈 0. 001 ）. Colony formation rate （84. 44% ）of 5-8F/shEZH2 cells was lower than control （31.56%, P =-0. 001 ）. Cell cycle analysis showed that most 5-8F/shEZH2 ceils were arrested in G0/G1 phase, with a very low ratio of cells in S phase. Wound healing assay indicated that the migration ability of cells silencing EZH2 decreased significantly, and the 48-hour relative migration distance of 5-8F/ShEZH2 cells and control cells was 0. 58 ± 0. 05, and 0. 8 1± 0. 02, respecptively （P 〈 0. 000）. Matrigel invasion assay, showed the invasive capacity of ceils silencing EZH2 was significantly inhibited, with less penetrating cells （72. 23 ±4. 08 ） compared to control（ 150.95 ±16.27）, P 〈 0. 000. The mRNA expressions of epithelial markers E-cadherin and Keratin 18 in the ceils silencing EZH2 increased by 177% and 158% respectively, and the mRNA expressions of mesenchymal markers β-catenin and N-cadherin decreased by 18.04% and 41.18% respectively. Similar results also were obtained with Western blot a

关 键 词：鼻咽肿瘤 DNA结合蛋白质类 转录因子 基因沉默 细胞增殖 肿瘤浸润 上皮间质转化 

分 类 号：R739.63[医药卫生—肿瘤]