CAV1基因沉默增强MCF-7多细胞球对多柔比星的敏感性  

CAV1 gene silencing enhances adriamycin sensitivity of breast carcinoma MCF-7 multicellular spheroids

作　　者：顾栋桦[1] 平金良[1] 董吉顺[1] 朱荣[2] 陈琦[2] 

机构地区：[1]浙江省湖州市中心医院病理科,浙江湖州313000 [2]复旦大学上海医学院病理学系,上海200032

出　　处：《中国癌症杂志》2011年第9期661-665,共5页China Oncology

基　　金：湖州市科技局科技攻关项目(No:2008GS07)

摘　　要：背景与目的:肿瘤细胞形成多细胞球后,对许多化疗药物的耐受性明显增强,这种现象被称为"多细胞耐药"。其发生机制可能与细胞黏附引起的信号通路活性的改变有关。细胞质膜微囊蛋白-1(caveolin-1,CAV1)是一个细胞膜蛋白,在细胞膜信号通路的调节中发挥重要作用。本研究旨在探讨CAV1基因沉默是否能增强MCF-7多细胞球对多柔比星的敏感性,并初步探讨其机制。方法:用liquid overlay技术培养获得MCF-7多细胞球,用脂质体转染法把特异针对CAV1基因的双链小RNA干扰片段导入MCF-7细胞中,采用台盼蓝拒染法检测多柔比星对MCF-7的细胞抑制率,分别用RT-PCR和免疫印迹法检测CAV1 mRNA水平及蛋白水平,用免疫印迹法检测细胞总黏着斑激酶(focal adhesion kinase,FAK)及磷酸化FAK(p-FAK)蛋白水平。结果:与单层细胞相比,多细胞球组的多柔比星细胞抑制率明显降低,FAK相对磷酸化水平增加2.64倍(t=7.32,P<0.01);CAV1基因沉默后,MCF-7多细胞球生长缓慢,球体较松散,与未转染组相比,干扰组多细胞球多柔比星(1μmol/L)的细胞抑制率增加了1.07倍(t=5.77,P<0.01),FAK相对磷酸化水平下降68.7%(t=6.57,P<0.01)。结论:多细胞球的形成可以增强MCF-7细胞对多柔比星的耐受性;CAV1基因沉默能逆转MCF-7多细胞球对多柔比星的耐药现象,FAK相对磷酸化水平的下调可能是其重要机制。Background and purpose：Tumor spheroids exhibit decreased sensitivity to anticancer drugs compared with monolayer cells,which is known as multicellular resistance（MCR）.Its mechanism underlying the phenomenon is unknown,but the signal transduction modified by cell-cell adhesion may be important in this procedure.Caveolin-1（CAV1） is a membrane protein which can regulate cell signal transduction.This study aimed to investigate the role of CAV1 gene expression on drug resistance of multicellular spheroids（MCS） of breast carcinoma to adriamycin（ADM） through the use of the RNA interfering technique.Methods：MCF-7 MCS were obtained from liquid overlay technique culture,and CAV1-targeted small interfering double-stranded RNAs（siRNA） were introduced into MCF-7 cells by LipofectamineTM2000.ADM resistance was detected with trypan blue exclusion testing.CAV1 mRNA and protein levels were determined by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot.The relative phosphorylation level of focal adhesion kinase（FAK） was assessed by Western blot.Results：Compared with monolayer cells,MCS showed lower cell inhibitory rate after ADM exposure for 24 h,and the relative phosphorylation level of FAK was elevated in MCS by 2.64-fold.CAV1-targeted RNA interference markedly inhibited the expression of CAV1 gene and suppressed formation of MCF-7 MCS.Cell inhibitory rate of ADM was enhanced by 1.07-fold and the relative phosphorylation level of FAK was reduced by 68.7% by CAV1 gene silencing in MCF-7 MCS.Conclusion：MCS cultures induce MCF-7 resistance to ADM.RNA interference to target CAV1 gene can restore sensitivity to ADM in MCS,and down-regulation of FAK phosphorylation may be involved in its mechanism.

关 键 词：质膜微囊蛋白 黏着斑激酶 球形体 细胞 抗药性 

分 类 号：R73-361[医药卫生—肿瘤]