抑制miR-30a/HMGA2介导的骨肉瘤细胞自噬对化学药物治疗诱导的细胞凋亡的作用  

Suppression of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells impacts chemotherapeutics-induced apoptosis

作　　者：夏勤 倪江东[1] 黄俊[1] 潘柏祺 严明明[1] 李文钊 XIA Qin;NI Jiangdong;HUANG Jun;PAN Baiqi;YAN Mingming;LI Wenzhao(Department of Orthopedics,Second Xiangya Hospital,Central South University,Changsha 410011,China)

机构地区：[1]中南大学湘雅二医院骨科

出　　处：《中南大学学报（医学版）》2019年第7期757-766,共10页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81302338);湖南省自然科学基金(2018JJ2567)~~

摘　　要：目的:探讨微小RNA(microRNA,miR)-30a/高迁移率族蛋白A2(high mobility group protein A2,HMGA2)介导的骨肉瘤细胞自噬对化学药物治疗(以下简称化疗)药物诱导的细胞凋亡的影响。方法:随机选取30例经化疗药物治疗后表现为化疗敏感和化疗抵抗的骨肉瘤患者组织,分为化疗敏感组(n=15)和化疗抵抗组(n=15),采用real-time PCR检测两组中miR-30a和HMGA2的mRNA表达水平,以及骨肉瘤细胞U2-OS经不同浓度的化疗药物(顺铂、阿霉素、氨甲蝶呤)处理后miR-30a的mRNA表达水平;采用蛋白质印迹法检测细胞内自噬相关因子Beclin 1,自噬微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B,LC3B)、自噬抑制因子P62的表达情况。在骨肉瘤细胞U2-OS中转染miR-30a模拟物和抑制剂,构建miR-30a高表达组、低表达组和对照组,采用蛋白质印迹法检测经过顺铂和阿霉素处理后上述3组细胞内Beclin 1,LC3B和P62的表达情况;采用单丹磺酰尸胺(monodansylcada,MDC)染色法检测细胞内自噬水平,ROS荧光探针-二氢乙啶(dihydroethidium,DHE)检测细胞内ROS水平,细胞计数试剂盒8(cell counting kit-8,CCK-8)检测细胞存活率,流式细胞术检测细胞凋亡程度,线粒体膜电位荧光探针JC-1检测细胞线粒体氧化损伤程度;采用双荧光素酶法检测miR-30a与HMGA2的相互作用,同时通过转染HMGA2模拟物和HMGA2-shRNA干扰质粒载体,构建HMGA2高表达组、低表达组和对照组,采用蛋白质印迹法检测经过顺铂和阿霉素处理后上述3组细胞内Beclin 1,LC3B和P62的表达情况。结果:化疗抵抗组中miR-30a的mRNA水平显著低于化疗敏感组(P<0.05),HMGA2的表达与miR-30a相反(均P<0.05)。在相对低浓度(5μml/L)的化疗药物刺激下,骨肉瘤细胞U2-OS内的miR-30a mRNA表达下调,Beclin 1和LC3B均显著上调(均P<0.01),P62显著下调(P<0.01)。在miR-30a高表达组中,与对照组相比,Beclin 1和LC3B的表达水平与miR-30a明显下降(P<0.05),P62的表达水Objective:To investigate the effect of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells on apoptosis induced by chemotherapeutics.Methods:A total of 30 osteosarcoma tissues of sensitive and resistant to chemotherapeutics were divided into a chemotherapy-sensitive group and a chemotherapy-resistant group.The mRNA expression levels of miR-30a and high mobility group protein A2(HMGA2)in the chemotherapysensitive group and the chemotherapy-resistant group,and the mRNA expression levels of miR-30a in osteosarcoma U2-OS cells treated by cisplatin,doxorubicin and methotrexate at different concentrations were detected by real-time PCR.The expression levels of autophagy related protein Beclin 1,microtubule associated protein 1 light chain 3B(LC3B)and autophagy factor P62 were detected by Western blotting.The osteosarcoma U2-OS cells were transfected with miR-30a mimics and miR-30a inhibitors to construct a miR-30a high expression group,a miR-30a low expression group and a control group.The expression levels of Beclin 1,LC3B and P62 in osteosarcoma U2-OS cells after treatment of cisplatin and doxorubicin in these 3 groups were detected by Western blotting;the level of autophagy was detected by monodansylcada(MDC)staining;the level of ROS was detected by dihydroethidium(DHE);the level of cell surviving rate was detected by cell counting kit-8(CCK-8);the level of apoptosis was detected by annexin APC/PI double staining;the level of mitochondria oxidative damage was detected by mitochondrial membrane potential assay kit with JC-1(JC-1 method).The interaction between miR-30a and HMGA2 was detected by dual luciferase reporter assay.The osteosarcoma U2-OS cells were transfected with HMGA2 mimics and HMGA2-shRNA to construct a high HMGA2 group,a low HMGA2 group,and a control group.The expression levels of Beclin 1,LC3B and P62 in osteosarcoma U2-OS cells after the treatment of cisplatin were detected by Western blotting.Results:The level of miR-30a in the chemotherapy-resistant tissues was significantly lower than that in t

关 键 词：微小RNA-30a 自噬 凋亡 骨肉瘤 

分 类 号：R73[医药卫生—肿瘤]