微小RNA-15a／16-1重组慢病毒对人鼻咽癌CNE-2Z细胞生物特性的影响  

Influence of recombinant lentiviral vector encoding miR-15a/16-1 in biological features of humannasopharyngeal carcinoma CNE-2Z cells

作　　者：张春鸿[1] 方潇碧[1] 李文峰[2] 施清圆[1] 吴丽萍[1] 陈晓云[1] 黄振校[3] 武鹏[1] 王珍珍[1] 廖志苏[1] 

机构地区：[1]温州医学院附属第一医院耳鼻咽喉科,325000 [2]温州医学院附属第一医院放化疗科,325000 [3]首都医科大学附属北京同仁医院耳鼻咽喉头颈外科

出　　处：《中华耳鼻咽喉头颈外科杂志》2013年第5期405-411,共7页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：2010年浙江省科技计划项目基金资助（2010C34005）

摘　　要：目的探讨微小RNA（microRNA，miR）-15a／16-1重组慢病毒对人鼻咽癌CNE-2Z细胞生物特性的影响，并探讨其可能机制。方法重组慢病毒和空载体慢病毒转染CNE一2Z细胞后筛选绿色荧光蛋白阳性细胞，实时定量PCR（RT-qPCR）检测miR-15a、miR-16-1表达水平；将实验分为对照组、转染组、放射线照射组、转染联合放射线照射组，四甲基偶氮唑蓝法分析各组细胞增殖情况；流式细胞术检测各组细胞凋亡情况；克隆形成实验分析对照组和转染组对放射线敏感性的变化；RT-qPCR、Westernblot法检测bel-2mRNA及其蛋白的表达水平；分光光度法检测Caspase-2、Caspase-3的活化程度。结果转染组miR-15a、miR-16-1相对表达量（x±s，下同）分别为524．80±40．79、466．11±40．96，同对照组比较，差异有统计学意义（t=494．611，f=386．840，P=0．000）；转染联合放射线照射组细胞增殖抑制最明显（F=424．255，P=0．000）。对照组、转染组、放射线照射组及转染联合放射线照射组的细胞凋亡率分别为（2．2±1．4）％、（9．64-0．8）％、（2．9±1．1）％、（18．6±0．7）％，差异有统计学意义（F=158．519，P=0．000）。转染组同等剂量下（2、4、6、8Gy）存活分数、平均致死剂量（meanlethaldose，Do）、准阈剂量（quasi-thresholddose，Dq）均低于对照组。二者放射增强比（sensitizationenhancementratios，SER）（Do）、SER（Dq）分别为1．473、1．581。转染组、放射线照射组、转染联合放射线照射组bcl-2mRNA表达相对量组间比较差异无统计学意义（P〉0．05）。转染联合放射线照射组bcl-2蛋白表达下降最明显，转染组次之；对照组、放射线照射组、转染组、转染联合放射线照射组的Caspase-2活化程度分别为0．12±0．01、0．24±0．04、0．35±0．02、0．44±0．04（F=115．500，P=0．000），Caspase-3的活化程度分别为0．13±0．01、0．27±0．0Objective To study the influence of recombinant lentiviral vector encoding miR-15a/ 16-1 on biological features of human nasopharyngeal carcinoma CNE-2Z cells and underlying mechanisms. Methods GFP-positive CNE-2Z cells transfected with recombinant lentiviral vector were selected. The experiment was divided into control group, transfected group, radiotherapy group, transfected-radiotherapy group. Cell proliferation was analyzed by MTT. Apoptosis was detected by flow cytometry. Radiotherapysensitivity of the cells in control group and transfected group was evaluated by colony forming experiment. The expressions of miR-15a, miR-16-1 and bcl-2 mRNA were detected by real-time quantitative polymerase chain reaction （RT-qPCR）. The expression of bcl-2 protein was detected by Western blot. The activation of Caspase-2 and Caspase-3 was evaluated by spectrophotometry. Results Relative expression quantities of miR-15a and miR-16-1 in infected group were 524.80 ± 40.79 （t = 494. 611, P = 0. 000） and 466. 11 ±40. 96 （ t = 386. 8, P = 0. 000）, respectively. The proliferation of the cells in transfected-radiotherapy group was the most obvious, followed by the cells in radiotherapy group and transfected group （ F = 424. 3, P = 0. 000 ）. The apoptosis rates of control group, transfected group, radiotherapy group and transfected- radiotherapy group were （2.2 ± 1.4）%, （9.6± 0.8）%, （2.9 ±1.1）%, and （18.6 ± 0.7）% respectively（ F = 158. 5, P =0.000）. Clonogenic assay showed that the values of SF2, Do （ 1. 473） and Dq （ 1. 581 ） in transfected group were lower than those in control group. The relative expression levels of bcl-2 mRNA in transfected group, radiotherapy group, and transfected-radiotherapy group had no significant difference （ P 〉 0. 05 ）. Decrease in the expression of bcl-2 protein in transfected-radiotherapy group was most significantly, followed by that in transfected group. The percentages of activated Caspase-2 in control group, radiotherapy group, transfected gr

关 键 词：鼻咽肿瘤 细胞系 肿瘤 原癌基因蛋白质C-BCL-2 辐射耐受性 微RNAS 

分 类 号：R73[医药卫生—肿瘤]