微小RNA-10b靶向蛋白去乙酰化酶4调控雌激素受体阳性MCF-7乳腺癌细胞他莫昔芬耐药性  

Regulation of tamoxifen resistance in estrogen receptor positive MCF -7 breast cancer cells by microRNA-10b targeting histone deacetylase 4

作　　者：张建祥[1] 马艳梅[1] 王芳[1] 李建华[1] Zhang Jianxiang;Ma Yanmei;Wang Fang;Li Jianhua(Department of General Surgery, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Chin)

机构地区：[1]郑州大学第一附属医院普通外科,450052

出　　处：《中华实验外科杂志》2018年第6期1009-1012,共4页Chinese Journal of Experimental Surgery

基　　金：河南省医学科技攻关计划项目（201602043）;河南省基础与前沿技术研究计划项目（162300410114）

摘　　要：目的探讨微小RNA（miRNA，miR）-10b靶向蛋白去乙酰化酶4（HDACA）调控雌激素受体阳性（ER＋）MCF-7乳腺癌细胞对他莫昔芬的耐药性。方法建立ER＋且他莫昔芬耐药的MCF-7细胞株MCF．．．7TR，实时定量反转录聚合酶链反应（RT-qPCR）法检测MCF-7细胞和MCF-7-TR细胞miR-10b表达水平。分别采用噻唑蓝（MTr）法和锥虫蓝法检测转染pre-miR-10b、miR-10b抑制物、siHDACA的MCF-7细胞、MCF-7-TR细胞在不同他莫昔芬浓度下（0、5、10、15、20、30μmol／L）的增殖能力和活性。采用Westernblot检测HDACA蛋白表达情况。结果以MCF-7乳腺癌细胞miR-10b表达水平和侵袭能力作为标准，MCF-7-TR细胞乳腺癌细胞miR-10b表达水平和侵袭能力较MCF-7乳腺癌细胞分别为7．2±1．1和5．3±1．3。随着他莫昔芬浓度增加，各细胞增殖率均呈下降趋势，其中以MCF-7细胞最为明显，其次为MCF-7-TR＋miR-10b抑制物＋siHDAC4细胞以及MCF-7＋pre-miR-10b＋HDACA细胞，而MCF-7-TR＋siHDACA细胞和MCF-7-TR细胞增殖率下降程度最低。MCF-7细胞和MCF-7-TR＋miR-10b抑制物细胞的迁移能力下降最多，而MCF-7＋pre-miR-10b＋他莫昔芬（10μmol／L）细胞和MCF-7-TR＋他莫昔芬（10μmol／L）细胞的迁移能力比较差异无统计学意义（t=1．116，P=0．272），但显著高于MCF-7细胞和MCF-7-TR＋miR．10b抑制物细胞（t=0．124、10．769、14．569、11．577，P均为0．000）。MCF-7＋pre-miR-10b细胞HDACA蛋白表达水平低于MCF-7细胞，MCF-7-TR细胞HDACA蛋白表达水平MCF-7-TR＋miR-10b抑制物细胞，MCF-7＋siHDAC4细胞HDAC4蛋白表达水平低于MCF-7细胞。结论miR-lOb-HDAC4信号通路可能作为乳腺癌他莫昔芬耐药的一种分子机制。Objective To investigate the effect of microRNA - 10b （miR - lOb） targeting histone deacetylase 4 （HDAC4） on the resistance of estrogen receptor positive （ ER ＋ ） MCF - 7 breast cancer cells to tamoxifen. Methods ER ＋ and tamoxifen resistant MCF - 7 cell line MCF - 7 - TR was established, and miR - lOb expression level in MCF - 7 cells and MCF - 7 - TR cells was detected by reverse transcription re- al- time quantitative polymerase chain reaction （Real- time PCR）. Methyl thiazol tetrazolium （MTr） and trypan blue were used to detect the proliferation and activity of MCF - 7 cells and MCF - 7 - TR cells trans- fected with pre - miR - lOb, miR - 10b inhibitor and siHDACA at different tamoxifen concentrations （0, 5, 10, 15, 20, 30 p.mol/L）, respectively. The expression of HDAC4 protein was detected by Western blotting. Results Taken expression level and invasive ability of miR - lOb in MCF - 7 breast cancer cells as the standard, the miR - 10b expression level and invasion ability of MCF - 7 - TR cell breast cancer cells were 7.2 ± 1.1 and 5.3 ± 1.3 respectively. With the increase of the concentration of tamoxifen, de- creased the rate of cell proliferation in MCF - 7 cells, which is most obvious, followed by MCF - 7 - TR ＋ miR- 10b inhibitor ＋ siHDAC4 and MCF- 7 ＋ pre- miR- 10h ＋ ttDAC4 cells, and the proliferation of MCF - 7 - TR ＋ siHDAC4 （：ells and MCF - 7 - TR cells decreased the rate of the lowest. The migration a- bility of MCF- 7 eells and MCF- 7 -TR ＋ miR- 10b （：ells of the inhibitor decreased most, MCF- 7 ＋ pre -miR - 10b ＋ and tamoxi%n （ lO ixmol/L） MCF -7 - TR ＋ cells and tamoxifen （ l0 Ixmol/L） had no significant difl＇erence in the ability of cell migration （ t = 1. 116, P = 0. 272 ） , but was significantly higher than that of MCF-7 cells and MCF-7-TR＋miR-10b cells （t=0.124, 10.769, 14. 569, 11.577, P =0. 000）. The expression level of HDAC4 protein in MCF-7 ＋ pre -miR- 10b cells was lower than that in MCF - 7 （：ells, MCF -

关 键 词：乳腺癌 雌激素受体阳性 他莫昔芬耐药 微小RNA-10b 蛋白去乙酰化酶4 

分 类 号：R737.9[医药卫生—肿瘤]