miRNA-34a靶向抑制高迁移率族蛋白B1逆转骨肉瘤细胞顺铂耐药性机制研究  

miRNA-34a reverses cisplatin resistance of osteosarcoma through targeted inhibition of high mobility group box 1

作　　者：关哲[1] 张晓娟[2] 郭蕊[3] Guan Zhe;Zhang Xiaojuan;Guo Rui(Department of Bone and Soft Tissue Oncology,Shanxi Provincial Cancer Hospital,Taiyuan 030013,China;Department of Radiology,Shanxi Provincial Cancer Hospital,Taiyuan 030013,China;Department of Basic Medicine,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西省肿瘤医院骨与软组织肿瘤科,太原030013 [2]山西省肿瘤医院放射科,太原030013 [3]山西医科大学基础医学院,太原030001

出　　处：《肿瘤研究与临床》2021年第10期754-759,共6页Cancer Research and Clinic

摘　　要：目的探讨miRNA-34a(miR-34a)靶向高迁移率族蛋白B1(HMGB1)逆转骨肉瘤细胞顺铂耐药的相关机制。方法采用剂量递增间歇作用的方法构建MG-63顺铂耐药细胞株(MG-63/DDP)。使用不同浓度顺铂(0、1、5、10、20、30 μg/ml)处理MG-63/DDP细胞, 采用CCK-8法检测细胞存活率。将MG-63/DDP细胞分别转染miR-34a过表达载体(miR-34a过表达载体组)及miR-34a空表达载体(miR-34a-NC组), 采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-34a的相对表达量;使用不同浓度顺铂(0、1、5、10、20、30 μg/ml)处理细胞, 采用CCK-8法检测细胞存活率, 流式细胞术检测细胞凋亡情况;采用双荧光素酶报告基因实验验证miR-34a是否调控HMGB1的表达, 蛋白质印迹法检测转染后细胞的HMGB1蛋白表达量。将MG-63/DDP细胞分别转染HMGB1基因沉默载体(si-HMGB1组)及其阴性对照载体(si-NC组), 采用蛋白质印迹法检测HMGB1蛋白表达量;CCK-8法检测细胞存活率。结果 MG-63/DDP细胞株构建成功。不同浓度顺铂作用后, MG-63/DDP细胞存活率均高于MG-63细胞(均P<0.01)。MG-63/DDP细胞和MG-63细胞对顺铂的半数抑制浓度(IC50)分别为25.80 μg/ml和0.47 μg/ml。MG-63/DDP细胞中miR-34a相对表达量低于MG-63细胞(0.46±0.04比1.02±0.05, t=15.14, P<0.01);与miR-34a-NC组相比, miR-34a过表达载体组MG-63/DDP细胞中miR-34a相对表达量增加(1.67±0.09比1.00±0.02, t=-12.58, P<0.01)。miR-34a过表达载体组、miR-34a-NC组细胞存活率随着顺铂浓度增加而降低, 5~30 μg/ml顺铂作用后, miR-34a过表达载体组细胞存活率均低于miR-34a-NC组(均P<0.01)。20 μg/ml顺铂处理24 h后, miR-34a-NC组和miR-34a过表达载体组MG-63/DDP细胞凋亡率分别为(25.1±1.7)%、(42.3±2.3)%, miR-34a过表达载体组MG-63/DDP细胞具有更高的凋亡率(P<0.01)。双荧光素酶报告基因实验结果显示, 与miR-34a-NC组相比, miR-34a过表达载体组能够抑制PGL3-野生型-HMGB1荧光素酶活性, 差异具有统计Objective To investigate the related mechanism of miRNA-34a(miR-34a)reverses cisplatin resistance of osteosarcoma through targeted inhibition of high mobility group box 1(HMGB1).Methods The MG-63 cisplatin-resistant cell line(MG-63/DDP)was constructed by using dose escalation and intermittent action,and then the successfully constructed MG-63/DDP cells were treated with different concentrations of cisplatin(0,1,5,10,20,30μg/ml),and CCK-8 method was used to detect cell survival rate.The MG-63/DDP cells were transfected respectively and then randomly divided into two groups:the miR-34a overexpression vector group and the miR-34a empty expression vector(miR-34a-NC)group.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression level of miR-34a.Transfected cells were treated with different concentrations of cisplatin(0,1,5,10,20,30μg/ml),and CCK-8 method was used to detect cell survival rate,flow cytometry was used to detect cell apoptosis.The dual luciferase reporter gene experiment was used to verify whether miR-34a regulated the expression of HMGB1,and Western blotting method was used to detect the HMGB1 protein expression level of the transfected cells.MG-63/DDP cells were transfected respectively and then randomly divided into two groups:HMGB1 gene silencing vector(si-HMGB1)group and its negative control vector(si-NC)group.Western blotting method was used to detect HMGB1 protein expression level and CCK-8 method was used to detect cell survival rate.Results The MG-63/DDP cell line was successfully constructed.The survival rate of MG-63/DDP cells was higher than that of MG-63 cells when cells were treated with different concentrations of cisplatin(all P<0.01),and half inhibitory concentration(IC50)value of MG-63/DDP cells and MG-63 cells on cisplatin was 25.80μg/ml and 0.47μg/ml,respectively.qRT-PCR results showed that the relative expression level of miR-34a in MG-63/DDP cells was lower than that in MG-63 cells(0.46±0.04 vs.1.02±0.05,t=15.14,P<0.01);compared with m

关 键 词：骨肉瘤 微RNAS 抗药性 肿瘤 高迁移率族蛋白质类 顺铂 

分 类 号：R738.1[医药卫生—肿瘤]