RNAi介导的IBP表达抑制对人乳腺癌MDA-MB-231细胞细胞骨架的影响  

Effects of RNAi-mediated IBP expression inhibition on cytoskeleton of human breast cancer MDA-MB-231 cells

作　　者：陈忠蛟[1] 李鹏[1] 杨明珍[1] 陈安[1] 胡川闽[1] 

机构地区：[1]第三军医大学医学检验系临床生物化学教研室,重庆400038

出　　处：《重庆医学》2013年第1期1-5,共5页Chongqing medicine

基　　金：国家自然科学基金资助项目(30901458);第三军医大学回国人员科研启动基金资助项目(2009XHG06)

摘　　要：目的研究干扰素调节因子4结合蛋白(IBP)的表达抑制对人乳腺癌MDA-MB-231细胞中微丝、微管的影响。方法设计并合成针对人IBP mRNA的RNA干扰(RNAi)序列,构建RNAi慢病毒表达载体,与包装质粒共转染人胚肾293FT细胞获得病毒颗粒,以病毒感染MDA-MB-231细胞。采用免疫印迹及实时聚合酶链反应(RT-PCR)确定能有效抑制IBP的RNAi序列,筛选出获得稳定感染的细胞株并进行微丝、微管染色。结果成功构建了IBP特异性的RNAi慢病毒表达载体和对照载体,对MDA-MB-231细胞IBP的沉默效率为69.9%;通过微丝、微管染色证实IBP表达下调后,细胞形态出现明显变化,细胞体增大,丝状伪足减少,片状伪足增多,微管排列紊乱。结论 IBP蛋白通过影响微管、微丝的排列而改变MDA-MB-231细胞生物学行为。Objective To study the effects of interferon regulatory factor-4 binding protein（IBP） expression inhibition on mi crofilament and microtubule of human breast cancer MDA-MB-231 ceils. Methods RNA interference（RNAi） sequences targeting human IBP mRNA were designed and synthesized. Lentiviral vectors expression RNAi were constructed and then were co-trans- fected with packaging plasmids into human embryonic kidney 293FT cells to obtain virus particles for infection of MDA-MB-231 cells. Western blot and real time-polymerase chain reaction（RT-PCR） were employed to confirm the sequences which could inhibit IBP effectively. Cells with stable infection were screened out and then their microfilament and microtubule were subjected to stai ning. Results Lentiviral vector expression IBP-specific RNAi and control vector were constructed successfully. IBP-silence efficien- cy in MDA-MB-231 cells was 69. 9%. Microfilament and mierotubule staining confirmed that IBP expression in cells were down- regulated. Obvious morphological changes of cells were observed, such as increasing cell body, decreasing filopodia, increasing lamel- lipodia and disorder of microtubule arrangement. Conclusion IBP protein alter the biological behavior of MDA MB-231 cells via in- fluencing the arrangement of microtubules and microfilaments.

关 键 词：转染 RNA干扰 慢病毒感染 细胞骨架 基因 IBP 

分 类 号：R737.9[医药卫生—肿瘤]