miR-490-3p逆转MCF-7/ADM细胞对阿霉素耐药性的影响  

Reversal effect of miR-490-3p on the resistance of MCF-7/ADM cells to adriamycin

作　　者：王晓先[1] 孔凡君[2] 李飞[3] 张硕[3] 

机构地区：[1]解放军456医院药械科,济南250031 [2]解放军456医院肿瘤科,250031 [3]解放军456医院药学部,250031

出　　处：《临床肿瘤学杂志》2015年第9期780-786,共7页Chinese Clinical Oncology

摘　　要：目的探讨miR-490-3p在阿霉素（ADM）耐药乳腺癌细胞系MCF-7/ADM中的表达情况及其对MCF-7/ADM细胞增殖、凋亡和ADM耐药的影响。方法以野生型人乳腺癌细胞系MCF-7及其耐药型细胞系MCF-7/ADM为研究对象,用实时荧光定量PCR（q PCR）比较两种细胞中miR-490-3p的表达水平,将miR-490-3p的过表达载体pc DNA3.1（＋）-miR-490-3p瞬时转染至MCF-7/ADM细胞（过表达组）后采用q PCR检测转染效果,同时设pc DNA3.1（-）空载体对照组和空白对照组;采用MTT法测定转染pc DNA3.1（＋）-miR-490-3p对各组MCF-7/ADM细胞增殖能力的影响,测定ADM对MCF-7/ADM细胞的增殖抑制率并计算半数抑制浓度（IC50）及逆转倍数以评价对ADM敏感性的变化;分别于瞬时转染48 h后用Annexin V/FITC流式细胞术检测各组MCF-7/ADM细胞的凋亡率,Western blotting检测耐药蛋白P-糖蛋白（P-gp）,乳腺癌耐药蛋白（BCRP）和多药耐药相关蛋白1（MRP1）的表达,荧光分光光度计检测胞内ADM药物浓度,流式细胞仪检测P-gp活性。结果MCF-7/ADM细胞中miR-490-3p的表达水平为0.24±0.07,显著低于野生型MCF-7细胞的1.02±0.03（P〈0.05）;过表达组瞬时转染24~96 h的miR-490-3p水平持续升高,均高于空载体对照组和空白对照组（P〈0.05）;过表达组的增殖抑制率随转染时间的延长而增加,转染24、48、72和96 h的增殖抑制率依次为（17.52±1.87）%、（31.67±2.79）%、（45.09±1.88）%和（61.82±2.52）%,高于空载体对照组（P〈0.05）;ADM对过表达组的IC50值为（11.27±2.34）μg/ml,低于空白对照组的和空载体对照组（P〈0.05）,且过表达组相对于空白对照组和空载体对照组的逆转倍数分别为3.35倍和3.39倍;与其余两组相比,过表达组的MCF-7/ADM细胞早、晚期凋亡率和细胞内药物浓度均升高,P-gp、BCRP和MRP1表达及P-gp活性均降低,差异均有统计学意义（P〈0.05）。结论上调miR-490-3p可逆转MCF-7/ADM细胞对ADM的耐药性,�Objective To investigate the expression of miR-490-3p in adriamycin（ ADM） resistant human breast cancer cell line MCF-7 / ADM and its effect on the cell proliferation,apoptosis and resistance to ADM of MCF-7 / ADM cell line. Methods Cultured wild-type MCF-7 human breast cancer cells and its resistant cells MCF-7 / ADM were selected as research objectives. The realtime fluorescent quantitative PCR（ q PCR） was used to compare the level of miR-490-3p in both cell lines. The recombinant plasmid pc DNA3. 1（ ＋）-miR-490-3p to over-express miR-490-3p was transiently transfected into MCF-7 / ADM cells（ over-expression group）and then the transfection effect was verified by q PCR. Meanwhile,the pc DNA3. 1（-） empty control group and blank control group were set up. MTT method was used to determine the effect of pc DNA3. 1（ ＋）-miR-490-3p on the proliferation of each group. The inhibitory rate of ADM on the proliferation of MCF-7 / ADM cells was determined,and the inhibition concentration（ IC50） and the reversal factor were calculated to evaluate the sensitivity to ADM. The cells in each group were collected at 48 h post-transfection for the detection of apoptotic rate by flow cytometry with Annexin FITC / PI double staining. Meanwhile,the expression of resistance proteins including P-glycoprotein（ P-gp）,breast cancer resistance protein（ BCRP） and multidrug resistance associated protein 1（ MRP1） were tested by Western blotting,intracellular ADM concentration was detected by fluorescence photometer and P-gp activity by flow cytometry. Results The level of miR-490-3p in MCF-7 / ADM cells was 0. 24 ± 0. 07,significantly lower than 1. 02 ± 0. 03 of wild type MCF-7 cells（ P 0. 05）. The levels of miR-490-3p in the over-expression group were higher than those in the other two groups during 24-96 h post-transfection（ P 0. 05）. The proliferation inhibition rates were（ 17. 52 ± 1. 87） %,（ 31. 67 ± 2. 79） %,（ 45. 09 ± 1. 88） %and（ 61. 82 ± 2. 52） % at 48,24,72 and

关 键 词：乳腺癌 miR-490-3p 阿霉素耐药 耐药蛋白 

分 类 号：R737.9[医药卫生—肿瘤]