Twist沉默介导的间充质-上皮转化对乳腺癌干细胞特性的影响  

Silencing Twist gene leads to mesenchymal-to-epithelial transition and loss of stemlike properties in breast cancer

作　　者：殷茜[1] 胡艺冰[2] 罗智勇[1] 覃吉超[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院甲乳外科,武汉430030 [2]华中科技大学同济医学院附属同济医院胃肠外科,武汉430030

出　　处：《中华实验外科杂志》2016年第10期2341-2343,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81272660）

摘　　要：目的 观察Twist沉默介导的间充质-上皮转化（MET）对乳腺癌干细胞特性的影响.方法 小干扰RNA（siRNA）转染乳腺癌MDA-MB-231细胞后,采用实时定量聚合酶链反应（Real-time PCR）和Western blot法检测细胞中Twist及MET相关分子[Slug、Snail以及波形蛋白（Vimentin）]的mRNA和蛋白的表达;显微镜观察Twist沉默后细胞的形态变化;流式细胞仪检测CD44＋ CD24细胞比例;体外球体形成实验检测细胞自我更新能力;体内有限稀释实验检测细胞启动肿瘤能力.结果 si-Twist组与对照组比较,Twist基因的mRNA（0.24±0.04,P＜0.01）和蛋白表达量明显降低;细胞表现为上皮样形态,排列规则、细胞间连接紧密、成团生长;间质相关分子Slug、Snail、Vimentin mRNA（0.25±0.05,P＜0.01;0.34 ±0.03,P＜0.01;0.44±0.04,P＜0.01）和蛋白表达量明显降低;上皮标志物E-钙黏蛋白（E-cadherin）mRNA（58.71±1.27,P＜0.01）和蛋白表达量明显升高;肿瘤干细胞的百分比明显降低[（63.30±0.80）％比（86.50±0.70）％,P＜0.01];球体形成能力明显降低[（46.67±2.52）个比（109.67±2.52）个,P＜0.01];肿瘤起始细胞数目明显减少（1/122 851比1/20 064,P＜0.01）.结论 MDA-MB-231细胞中Twist沉默后,细胞发生MET,体外自我更新和体内成瘤能力下降.Objective To observe the effect of silencing Twist gene on mesenchymal-to-epithelial transition （MET） and stem-like properties in breast cancer.Methods The small interfering RNA （siRNA） vectors targeting Twist gene were transfected into MDA-MB-231 cells.The mRNA and protein expression levels of Twist and MET-associated genes （Slug,Snail and Vimentin） were detected by using real-time quantitative polymerase chain reaction（Real-time PCR） and Western blotting.Cell morphology was observed under microscope.The proportion of CD44 ＋ CD24-cells was measured by flow cytometry.In vitro self-renewal capacity was evaluated by sphere formation assay.Tumor-initiating ability was measured by limited dilution assays.Results Compared to the control group,the Twist mRNA （0.24 ± 0.04,P 〈 0.01） and protein expression in siRNA group was significantly decreased.Cells with Twist silencing underwent MET reversal,showing an epithelial-like appearance,decreased mRNA expression of mesenchymal markers,including Slug （0.25 ±0.05,P 〈0.01）,Snail （0.34 ±0.03,P 〈0.01）and Vimentin （0.44 ± 0.04,P 〈 0.01）,increased mRNA expression of epithelial marker,E-cadherin （58.71 ±1.27,P 〈 0.01）,down-regulated protein levels of Slug,Snail and Vimentin,and up-regulated protein level of E-cadherin.The proportion of CD44 ＋ CD24-cells in the si-Twist group and control group was （63.30 ± 0.80） % and （86.50 ± 0.70） % respectively （P 〈 0.01）.The sphere number in the si-Twist and control groups was （46.67 ±2.52） and （109.67 ±2.52） respectively （P〈0.01）.Twist silencing led to the decrease of about 6-folds in tumor-initiating ability in nude mice compared to the control （P 〈 0.01）.Conclusion Silencing Twist gene leads to MET and loss of self-renewal capacity and tumor-initiating ability in breast cancer.

关 键 词：乳腺癌 TWIST 小干扰RNA 间充质-上皮转化 肿瘤干细胞 

分 类 号：R737.9[医药卫生—肿瘤]