HMGB1/AMPK/m-TOR信号通路对K562细胞自噬和化学治疗耐药的影响  

Influence of HMGB1/MAPK/m-TOR signaling pathway on cell autophagy and chemotherapy resistance in K562 cells

作　　者：刘丽英[1] 高飞[1] 叶艳琼 陈志衡[3] 戴云鹏[1] 赵平[1] 管国涛[1] 赵明一[3] 

机构地区：[1]山东大学附属省立医院儿科,济南250021 [2]广东省人民医院心外科,广州510010 [3]中南大学湘雅三医院儿科,长沙410013

出　　处：《中南大学学报（医学版）》2016年第10期1016-1023,共8页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81200378,81500231);湖南省自然科学基金(2013JJ6018,2015JJ6118)~~

摘　　要：目的:观察高迁移率族蛋白1(high-mobility group box 1,HMGB1)对急性髓系白血病细胞株(human leukemiacell line,K562)自噬及化学治疗(化疗)耐药的影响,并探讨其相关的分子机制。方法:体外培养K562细胞,分为化疗药物处理组、化疗药物处理对照组、HMGB1纯化蛋白预处理组、HMGB1纯化蛋白预处理对照组、HMGB1si RNA转染组和HMGB1 si RNA转染对照组,其中化疗药物处理组又分为长春新碱(v incristine,VCR)、足叶乙甙(etoposide,VP-16)、阿糖胞苷(cytosine arabinoside,Ara-C)、阿霉素(adriamycin,ADM)和三氧化二砷(arsenic trioxide,As2O3)处理组。细胞计数试剂盒-8检测细胞活性;Western印迹检测HMGB1,微管相关蛋白1轻链3(microtubule-associate protein1light chain3,LC3),腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)和哺乳动物雷帕霉素靶点(mammalian target of rapamycin,m-TOR)磷酸化蛋白表达水平;ELISA法检测细胞上清液HMGB1蛋白含量;单丹磺酰尸胺染色和透射电镜观察细胞自噬状态。结果:与相应对照组比较,各化疗药物处理组(VCR,VP-16,Ara-C,ADM和As2O3)的细胞活性均显著降低,HMGB1蛋白表达均显著上调(均P<0.05)。与相应对照组比较,HMGB1纯化蛋白预处理组的细胞活性显著增加(P<0.05),HMGB1蛋白表达显著上调(P<0.05)。与HMGB si RNA转染对照组比较,HMGB1si RNA转染组的细胞活性显著降低(P<0.05),HMGB1蛋白表达显著下调(P<0.05);与相应对照组比较,HMGB1纯化蛋白处理组中LC3-II表达明显增强(P<0.05),光镜下观察到自噬小体和自噬泡数量明显增加。同时,与相应对照组比较,HMGB1纯化蛋白处理组p-AMPKa的蛋白表达明显增强,而p-m TOR表达明显减弱(均P<0.05)。结论:HMGB1可能通过AMPK/m-TOR信号通路增强K562细胞自噬发挥化疗耐药性。Objective： To observe the effect of high-mobility group box 1 （HMGB1） on autophagy and chemotherapy resistance in human leukemiacell line （K562） cells, and to explore the underlying mechanisms. Methods： The K562 cells were cultured in vitro and divided into 6 groups： a chemotherapeutic group, a chemotherapeutic control group, a HMGB 1 preconditioning group, a HMGB1 preconditioning control group, a HMGB1 siRNA group and a siRNA control group. The chemotherapeutic group was further divided into a vincristine （VCR） group, an etoposide （VP- 16） group, a cytosine arabinoside （Ara-C） group, a adriamycin （ADM） group and a arsenic trioxide （As203） group. The cell activity was evaluated by cell counting kit-8. The protein levels of HMGB1, microtubule-associate proteinllight chain3 （LC3）, AMP-activated protein kinase （AMPK） and mammalian target of rapamycin （m-TOR） were determined by Western blotting. The level of serum HMGB 1 was evaluated by enzyme-linked immunosorbent assay （ELISA）. The autophagy was examined by monodansylcadaverine staining and observed under transmission electron microscopy. Results： Compared with the control group, the cell activity was significantly decreased and the level of serum HMGB1 was significantly increased in the chemotherapeutic （VCR, VP-16, Ara-C, ADM and As203） groups （all P〈0.05）. Compared with the control group, the cell activity and the level of serum HMGB 1 were significantly increased in the HMGB 1 preconditioning group （both P〈0.05）. Compared with the siRNA control group, the cell activity and the level of serum HMGB1 were significantly decreased in the HMGB1 siRNA group （both P〈0.05）. Compared with the control group, the expression of LC3-Ⅱ and the formation of autophagic bodies were increased in the HMGB1 preconditioning group （both P〈0.05）, the p-AMPK expression was increased and p-mTOR expression was decreased （both P〈0.05）. Conclusion： HMGB1 can increase the autophagy and promote chemothe

关 键 词：儿童白血病 高迁移率族蛋白1 自噬 化学治疗耐药 腺苷酸活化蛋白激酶 哺乳动物雷帕霉素靶蛋白 

分 类 号：R733.71[医药卫生—肿瘤]