CD44v16通过细胞自噬途径增强乳腺癌MCF7细胞的化疗耐药性  

CD44v16 contributes to chemoresistance via the modulation of autophagy in breast cancer MCF7 cells

作　　者：钱傅燕雯 冯凡[1,2] 许文林 QIAN Fuyanwen;FENG Fan;XU Wenlin(Central Laboratory,Fourth People's Hospital Affiliated to Jiangsu University,Zhenjiang,212001,Jiangsu Province,China;Clinical Medicine Research Office,Medicine School of Jiangsu University,Zhenjiang,212013,Jiangsu Province,China)

机构地区：[1]江苏大学附属第四人民医院中心实验室,江苏缜江212001 [2]江苏大学医学院临床医学研究室,江苏镇江212013

出　　处：《肿瘤》2019年第6期439-449,共11页Tumor

基　　金：国家自然科学基金面上项目(编号:81672913)~~

摘　　要：目的:探讨一种新的CD44剪切变异体16(CD44 variant 16,CD44v16)对乳腺癌细胞多柔比星(doxorubicin,DOX)耐药性的影响及其机制。方法:利用慢病毒感染法构建CD44v16过表达的MCF7细胞株(命名为MCF7-CD44v16),以及CD 44v 16基因沉默的MCF7/DOX细胞株(命名为MCF7/DOX-sh CD44v16)。采用CCK-8法检测DOX对各组细胞的半数抑制浓度(half maximal inhibitory concentration,IC50),以及在相同浓度DOX处理后细胞的生长情况。采用罗丹明潴留实验检测各组细胞中罗丹明潴留率。采用实时荧光定量PCR法检测各组细胞中乳腺癌耐药相关基因的表达变化。采用蛋白质印迹法检测各组细胞中自噬相关蛋白的表达水平。用自噬抑制剂氯喹(30nmol/L)处理各组细胞后,再次采用CCK-8和实时荧光定量PCR法分别检测各组细胞的DOX IC50值和耐药相关基因的表达变化。结果:成功构建慢病毒稳定感染的MCF7-CD44v16和MCF7/DOX-sh CD44v16细胞株。DOX对各组细胞的IC50值存在明显差异(P<0.05)。DOX作用下MCF7-CD44v16细胞生长活性较MCF7细胞高(P<0.05),相反MCF7/DOX-shCD44v16细胞生长活性较MCF7/DOX细胞低(P<0.05)。MCF7-CD44v16细胞中罗丹明平均荧光强度低于MCF7细胞(P<0.05),而MCF7/DOX-shCD44v16细胞中罗丹明潴留率高于MCF7/DOX细胞(P<0.05)。与对照组MCF7细胞相比,MCF7-CD44v16细胞中CD44v16过表达,多药耐药相关蛋白1(multidrug resistance-associated protein 1,MRP1)、肺耐药蛋白(lung resistance protein,LRP)和切除修复交叉互补基因1(excision repair cross-complementing gene 1,ERCC1)的mRNA表达水平明显上调(P值均<0.01),微管相关蛋白1轻链3Ⅱ(microtubule-associated protein 1 light chain 3Ⅱ,LC3Ⅱ)及Bcl-2同源结构域蛋白1(Beclin-1)表达水平也明显升高(P值均<0.01);而与对照组MCF7/DOX细胞株相比,MCF7/DOX-shCD44v16细胞株中CD44v16表达下调后,MRP1、LRP、ERCC1、谷胱甘肽-S-转移酶π亚型(glutathione-S-transporter-π,GST-π)及多药耐药基因1(multidrug rObjective:To explore the effect of a new CD44 variant 16(CD44v16)on doxorubicin(DOX)resistance in breast cancer cells and its mechanism.Methods:CD44v16-overexpressed MCF7 cell line MCF7-CD44v16 and CD44v16 genesilenced MCF7/DOX cell line MCF7/DOX-shCD44v16 were constructed by lentivirus infection.CCK-8 method was used to detect the half maximal inhibitory concentration(IC50)of DOX in various cells and the growth of cells treated with DOX at the same concentration.Rhodamine retention rate was detected by rhodamine retention experiment.The expressions of various breast cancer resistance-related genes in various cells were detected by real-time fluorescent quantitative PCR.The expressions of autophagy-related proteins were detected by Western blotting.After the treatment of autophagy inhibitor chloroquine(30 nmol/L),the IC50 value of DOX and the changes of drug resistance-related gene expressions in various cells were detected again by CCK-8 and real-time fluorescent quantitative PCR,respectively.Results:MCF7-CD44v16 and MCF7/DOX-shCD44v16 cell lines were successfully constructed.The IC50 values of DOX in MCF7,MCF7-CD44v16,MCF7/DOX and MCF7/DOX-shCD44v16 cells had significant differences(P<0.05).The growth ability of MCF7-CD44v16 cells was higher than that of MCF7 cells treated with DOX(P<0.05),while the growth ability of MCF7/DOXshCD44 v16 cells was lower than that of MCF7/DOX cells(P<0.05).The mean fluorescence intensity of rhodamine in MCF7-CD44v16 cells was lower than that in MCF7 cells(P<0.05),while the retention rate of rhodamine in MCF7/DOX-shCD44 v16 cells was higher than that in MCF7/DOX cells(P<0.05).Compared with MCF7 cells,CD44v16 was overexpressed in MCF7-CD44v16 cells,and the expression levels of multidrug resistance-associated protein 1(MRP1),lung resistance protein(LRP),excision repair cross-complementing gene 1(ERCC1)mRNAs were significantly up-regulated(all P<0.01),while the expression levels of microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ)and Bcl-2 homologous domain protein-1(Beclin-1)

关 键 词：乳腺肿瘤 抗药性 肿瘤 自噬 多药耐药相关蛋白质类 CD44v16 

分 类 号：R737.9[医药卫生—肿瘤]