摘要
目的探讨微小RNA（miRNA，miR）-19a通过乳腺癌阿霉素（ADM）耐药癌株MCF-7/A来源的外泌体在化疗药物耐药性传递中的作用及其潜在的分子机制。方法采用超速分级离心法从乳腺癌细胞培养上清中分离提取外泌体，投射电镜及Nanosight对外泌体进行观察鉴定。利用重组慢病毒载体构建稳定表达绿色荧光蛋白（GFP）的乳腺癌敏感细胞株GFP-MCF-7/S，通过细胞-细胞及细胞-外泌体共培养实验观察耐药性传递现象。实时定量聚合酶链反应（Real-time PCR）、Western blot及细胞转染技术检测miR-19a及第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因/蛋白激酶B/p53（PTEN/Akt/p53）轴在耐药性发生过程中的作用。结果与MCF-7/S比较，MCF-7/A细胞中miR-19a的表达（1.03±0.16比3.56±0.58，P=0.002）显著增加，miR-19a抑制剂处理后MCF-7/A细胞对化疗药物ADM的敏感性[（41.17±10.65）%比（23.45±8.89）%，P=0.011]显著增加。电镜下观察两种乳腺癌细胞分泌的外泌体均呈圆形或椭圆形，直径为40～100 nm。荧光显微镜下对GFP的观察及细胞计数试剂盒（CCK-8）法对细胞增殖抑制率的检测结果显示ADM对MCF-7/S ＋MCF-7/A及MCF-7/S＋ MCF-7/A exos组细胞的增殖抑制率显著低于MCF-7/S＋MCF-7/S[（31.50±6.68）%比（51.20±12.44）%，P=0.006]及MCF-7/S＋ MCF-7/S exos[（27.67±6.02）%比（49.65±10.52）%，P=0.000]组细胞，且伴随着细胞miR-19a表达的增加（1.01±0.13比2.67±0.62，P=0.000）。Western blot检测结果显示外泌体中miR-19a能够通过显著抑制PTEN（1.15±0.18比0.67±0.13，P=0.000）及p53（0.99±0.18比0.49±0.11，P=0.005）蛋白的表达，增加Akt的磷酸化水平（1.15±0.18比0.67±0.13，P=0.000）促进受体细胞耐药的发生。结论miR-19a可通过外泌体包裹的形式传递乳腺癌细胞间的耐药性，进而通过对PTEN/Akt/p53轴的调控促进受体细胞耐药的发生。
ObjectiveTo investigate the specific effect and underlying molecular mechansim of microRNA （miRNA, miR）-19a through MCF-7/A secrete dexosomes in drug resistance transfer.MethodsExosomes were extracted from the supernatant of cultured MCF-7/A cells by fractionation ultracentrifugation and were identified by transmission electron microscopy and Nanosight. GFP-MCF-7/S, a breast cancer sensitive cell line stably expressing green fluorescent protein （GFP）, was constructed by recombinant lentiviral vector with GFP. The co-cultured experiments of cell-cell and cell-exosomewere designed to observe thephenomenon of drug resistance transfer. Real-time quantitative polymerase chain reaction（Real-time PCR）, Western blotting and cell transfection techniquewere used to determine the specific effects of miR-19a and phosphatase and tensin homologue deleted on chromosometen （PTEN）/protein kinase B （Akt）/p53 axis in the progress of drug resistance.ResultsCompared to MCF-7/S, the expression of miR-19a was significantly increased in MCF-7/A cells （1.03±0.16 vs. 3.56±0.58, P=0.002）, down-regulaion of miR-19a apparently increased sensitivity to Adriamycin （ADM） in MCF-7/A cells [（41.17±10.65）% vs. （23.45±8.89）%, P=0.011]. Transmission electron microscopy showed that the exosomes cells had adiameter of 40-100 nm and exhibited round or elliptic shape secreted by both two type cancer cells. The cell inhibition ratio of ADM detected by the intensity of GFP and cell counting kit-8 （CCK-8） assay showed that cell inhibition rate of MCF-7/S＋ MCF-7/A and MCF-7/S＋ MCF-7/A exos group were significantly lower than the MCF-7/S＋ MCF-7/S [（31.50±6.68）% vs. （51.20±12.44）%, P=0.006] and MCF-7/S＋ MCF-7/S exos group [（27.67±6.02）% vs. （49.65±10.52）%, P=0.000], which companied with the increasing expression miR-19a （1.01±0.13 vs. 2.67±0.62, P=0.000）. Western blotting assay showed that miR-19 in exosomes secreted by drug resistant cancer cells significantly inhibited the express
出处
《中华实验外科杂志》
CSCD
北大核心
2017年第5期770-773,共4页
Chinese Journal of Experimental Surgery
基金
河南省教育厅科学技术研究重点项目（13A360610）
