下调Oct4基因表达对MDA—MB-231乳腺癌干细胞生物学特性的影响  

Effect of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells

作　　者：李文鹏[1] 罗维远[1] 徐毅[2] 丁伟基 陈跃达[2] 张传凯[1] 罗琪[1] 黄正接[1] 

机构地区：[1]厦门大学附属第一医院厦门市肿瘤中心肿瘤外科,361003 [2]福建医科大学第一临床医学院

出　　处：《中华肿瘤杂志》2015年第4期251-257,共7页Chinese Journal of Oncology

基　　金：福建省医学创新课题（2012-CXB-29）;厦门市科技计划项目（3502220134011、3502220124018）

摘　　要：目的探讨下调转录因子Oct4表达水平对MDA-MB-231乳腺癌干细胞生物学特性的影响及其意义。方法应用无血清悬浮培养法从乳腺癌MDA-MB-231细胞中分离富集乳腺癌干细胞，流式细胞术检测CD44^＋／CD24^-1/low细胞亚群的比例，小鼠皮下成瘤实验和紫杉醇抑制实验鉴定无血清悬浮培养法获得的MDA—MB-231乳腺癌干细胞，逆转录聚合酶链反应和Western blot法检测MDA—MB-231乳腺癌干细胞中Oct4的表达情况，并利用小分子RNA干扰片段干扰Oct4的表达，干扰实验设空白对照组（不转染任何siRNA）、阴性对照组（转染对细胞无任何干扰作用的siRNA序列）和Oct4siRNA组（转染对Oct4基因有干扰作用的siRNA序列）。分别利用四甲基偶氮唑蓝法、Transwell小室法和紫杉醇抑制实验，检测干扰Oct4表达后MDA—MB-231乳腺癌干细胞的增殖能力、侵袭能力和对化疗药物的耐药性。结果MDA—MB-231乳腺癌于细胞以悬浮球的形式生长扩增，MDA—MB-231乳腺癌干细胞CD44^＋／CD24^-/low细胞亚群的比例（92．7％）高于MDA—MB-231乳腺癌细胞（76．6％，P〈0．05）。接种0．2×10^7个／ml细胞数，MDA—MB-231乳腺癌干细胞肿瘤体积为（124．60±13．65）mm^3，大于MDA-MB-231乳腺癌细胞形成的肿瘤体积[（68．20±9．99）mm^3，P〈0．01）]。紫杉醇对MDA-MB-231乳腺癌干细胞的半数抑制浓度（IC50）为（8．20±0．34）μg／ml，高于MDA—MB-231乳腺癌细胞[（4．40±0．48）μg／ml，P〈0．01）]。MDA-MB-231乳腺癌干细胞Oct4的表达水平高于乳腺癌细胞（P〈0．05）。下调Oct4蛋白的表达后，Oct4siRNA组MDA—MB-231乳腺癌干细胞从第3天开始其增殖能力低于空白对照组和阴性对照组（均P〈0．05）。侵袭性实验结果显示，转染72h后，与空白对照组和阴性对照组比较，Oct4siRNA组MDA—MB-231乳腺癌于细胞穿膜数量为（46．52±2．58）个，少于空白对照组[（77．29±Objective To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells. Methods Breast cancer cell line MDA- MB-231 cells were used in this study. Breast cancer stem cells were isolated and enriched by serum-free culture. The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice. RT-PCR, realtime PCR （qPCR） and Western blot were used to detect Oct4 expression. RNA interference was applied to induce Oct4 down-regulation. The interference experiment set up a control group （no siRNA transfection） ,negative control group （ negative siRNA group, transfection of siRNA sequences without any interfering effect on the cells） and Oct4 siRNA group （transfection of siRNA with interfering effect on the Oct4 gene）. Methyl thiazolyl tetrazolium （MTr） and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Results MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension. MDA- MB-231 breast cancer stem cells showed a higher percentage of CDd4^＋/CD24^-/low cells （97.2%） than that in MDA-MB-231 breast cancer cells （76.6%） （P〈0.05）. The tumor size in mice inoculated with MDA-MB-231 breast cancer stem ceUs was （124.60±15.65）mm^3, significantly larger than that of mice inoculated with breast cancer cells （68.20±9.99 mm^3） （P=0.0007）. MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration （IC50） [ （4.40±0.48） μg/ml vs. （8.20±0.34） μg/ml, P〈0.05]. However, the expression of transcriptional factors Oct4 wa

关 键 词：乳腺肿瘤 转录因子 肿瘤干细胞 悬浮培养 RNA干扰 小鼠 

分 类 号：R737.9[医药卫生—肿瘤]