沉默Bmi-1基因对CD44^+鼻咽癌类肿瘤干细胞增殖调控的影响  

Effect of gene silencing of Bmi-1 on proliferation regulation of CD44^+ nasopharyngeal carcinoma cancer stem-like cells

作　　者：许新华[1,2] 刘洋[1,2] 李道俊[3] 苏进[3] 胡娟[3] 鲁明骞[1,2] 易芳[3] 任精华[4] 陈卫红[4] 

机构地区：[1]三峡大学第一临床医学院 [2]宜昌中心人民医院肿瘤科,湖北宜昌443003 [3]三峡大学肿瘤研究所 [4]华中科技大学同济医学院附属协和医院肿瘤中心

出　　处：《临床耳鼻咽喉头颈外科杂志》2015年第10期941-947,共7页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：湖北省卫生厅科研项目(No:JX4B52);2011年宜昌市科技研究与开发项目(No:A11301-04);2011年度湖北省自然科学基金项目(No:2011CDB330)

摘　　要：目的:探讨应用RNAi沉默Bmi-1基因对CD44+鼻咽癌类肿瘤干细胞增殖调控的影响。方法:构建靶向Bmi-1基因的短发卡RNA慢病毒(LV-Bmi-1shRNA),感染通过流式细胞仪分选的CD44+鼻咽癌细胞;利用荧光显微镜及流式细胞仪检测感染效率;Real-time PCR及Western blot方法检测Bmi-1及其下游基因mRNA与蛋白表达;CCK-8增殖实验检测细胞增殖;成球实验检测成球能力;平板克隆形成实验检测克隆形成能力;流式细胞仪分析各组细胞周期分布情况。结果:LV-Bmi-1shRNA感染效率达95%以上;与空载体组和空白对照组相比,实验组Bmi-1mRNA及蛋白表达明显下调(P<0.05),而其下游基因p16INK4a、p14ARF mRNA及蛋白则上调;实验组的增殖、克隆形成及成球能力均明显降低;G0/G1期细胞比率增加,G2/M及S期比率降低。结论:Bmi-1基因沉默可抑制CD44+鼻咽癌类肿瘤干细胞的增殖、克隆形成及自我更新能力,使细胞周期发生阻滞,其作用可能是通过Bmi-1-p16INK4a/p14ARF-p53通路介导的。表明Bmi-1基因可能对CD44+鼻咽癌细胞干性样特征的维持有重要作用,并由此可能成为鼻咽癌治疗的一个潜在新靶点。Abstract Objective：To investigate the effect of gene silencing of Bmi-1 on proliferation regulation of CD44＋ nasopharyngeal carcinoma cancer stem-like cells （CSC-LCs）. Method： The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 gene （LV-Bmi-lshRNA） was constructed and was used to infect CD44＋ naso- pharyngeal carcinoma cells which were sorted by flow cytometry. A lentiviral which included a random sequence was also designed to serve as a negative control. We employed fluorescence microscope and flow cytometry to de- tect infection effieiency~ real-time PCR was used to detect Bmi-1 and its downstream gene while each protein ex- pression level was confirmed by western blotting protocols CCK-8 proliferation assay was applied to measure pro- liferation capacity tumor spheroid assay was used to evaluate the self-renewal capacity. Colony formation assay was used to measure cell colony formation capability flow cytometry analyzed cell cycle distribution. Result： The constructed LV-Bmi-lshRNA successfully infected into the CD44＋ nasopharyngeal carcinoma ceils. , The infection efficiency could reach above 95% LV-Bmi-lshRNA effectively inhibited Bmi-1 mRNA and protein expression, while the downstream gene p16INK4 and p14ARe mRNA as well as protein expression level were upregulated （P〈 0. 05）. Notablely, the proliferation, colony formation, self-renewal capabilities of the experimental group de- creased significantly （P〈0.05）. In addition, the cell cycle arrested at the G0-G1 phase. Conclusion：Gene silencing of Bmi-1 inhibited the proliferation, colony formation and self-renewal capabilities of the CD44＋ nasopharyngeal carcinoma CSC-LCs, inhibited the cell cycle processes, which may mediate through Bmil-p16INK4a/p14ARV-p53 pathway. Our experimental results indicated that Bmi-1 gene may play an important role in the maintenance of the stem cell-like characteristics of CD44＋ nasopharyngeal carcinoma cells. Bmi-1 gene may be a potential new target for the treatment

关 键 词：BMI-1基因 CD44 鼻咽癌 类肿瘤干细胞 

分 类 号：R739.6[医药卫生—肿瘤]