BRD4/NF-κB信号通路介导的铁死亡参与三阴性乳腺癌化疗耐药的机制  

Mechanism of ferroptosis mediated by the bromodomain protein subfamily 4/nuclear factor-kappa B signaling pathway in chemotherapy resistance of triple-negative breast cancer

作　　者：张硕稳 李丹[1] 贺静[1] 杜志兴[1] 裴永彬[2] Zhang Shuowen;Li Dan;He Jing;Du Zhixing;Pei Yongbin(Health Management Center,The First Hospital of Hebei Medical University;Department of General Surgery,The First Hospital of Hebei Medical University)

机构地区：[1]河北医科大学第一医院健康管理中心,石家庄050000 [2]河北医科大学第一医院普外科,石家庄050000

出　　处：《重庆医科大学学报》2024年第7期844-852,共9页Journal of Chongqing Medical University

基　　金：河北省卫生健康委科研基金资助项目(编号:20190484)。

摘　　要：目的:探讨溴域蛋白亚家族4(bromodomain protein subfamily 4,BRD4)/核因子κB(nuclear factor kappa-B,NF-κB)信号通路介导的铁死亡参与乳腺癌恶性生物学行为的机制。方法:多柔比星(Doxorubicin,DOX)抗性MDA-MB-231细胞(MDAMB-231/DOX)分为对照(Con)组、DOX组和si-BRD4+DOX组。si-BRD4+DOX组在用si-BRD4转染MDA-MB-231/DOX细胞48 h后,用1μmol/L DOX处理细胞24 h。通过集落形成试验和5-乙炔基-2′-脱氧尿苷(5-Ethynyl-2′-deoxyuridine,EdU)分析评估细胞增殖能力。通过FerroOrange、liperfloo检测细胞亚铁离子浓度和脂质过氧化水平。将15只雌性BALB/c-nu小鼠随机分为3组:对照组、DOX组、DOX+si-BRD4组,每组5只,用于建立MDA-MB-231/DOX细胞皮下接种模型。通过qRT-PCR、蛋白质印迹、免疫组化分析BRD4/NF-κB信号通路表达。结果:与si-NC组相比,si-BRD4#1和si-BRD4#2组MDA-MB-231、BT549细胞的细胞克隆数、EDU阳性染色、谷胱甘肽(glutathione,GSH)水平均降低(P<0.001),和MDA-MB-231、BT549细胞亚铁离子水平、活性氧(reactive oxygen species,ROS)水平、氧化型谷胱甘肽(Oxidized glutathione,GSSG)/GSH比值升高(P<0.01)。与亲代细胞(MDA-MB-231)相比,在MDA-MB-231/DOX中BRD4的mRNA和蛋白质表达水平升高。与Con组相比,DOX组细胞中IKβ-α、NF-κB、BRD4表达和ROS水平增加(P<0.05),和细胞克隆数和EDU阳性染色均降低(P<0.05);与DOX组相比,si-BRD4+DOX组细胞中核因子κB抑制蛋白α(Anti-IKB alpha,IKβ-α)、NF-κB表达、细胞克隆数、EDU阳性染色降低(P<0.05),ROS水平升高(P<0.05)。与对照组相比,DOX组肿瘤重量和体积均减少(P<0.05),并且肿瘤组织中TUNEL染色细胞增加(P<0.05)。此外,BRD4+DOX组肿瘤重量和体积较DOX组进一步降低(P<0.001),TUNEL染色细胞进一步增加(P<0.001)。BRD4+DOX组肿瘤组织中Ki-67、BRD4、NF-κB较DOX组下调(P<0.01),4-羟基壬烯醛(4-Hydroxynonenal,4-HNE)上调(P<0.01)。结论:BRD4是一个重要的耐药因子,它可以通过促进NF-κB信号�Objective:To investigate the mechanism of ferroptosis mediated by the bromodomain protein subfamily 4(BRD4)/nuclear factor-kappa B(NF-κB)signaling pathway in the malignant biological behavior of breast cancer.Methods:Doxorubicin-resistant MDA-MB-231 cells(MDA-MB-231/DOX)were divided into control group(Con group),DOX group,and si-BRD4+DOX group.In the si-BRD4+DOX group,MDA-MB-231/DOX cells were transfected with si-BRD4 for 48 hours and were then treated with 1μmol/L DOX for 24 hours.Colony formation assay and 5-ethynyl-2'-deoxy⁃uridine(EdU)analysis were used to evaluate the proliferation ability of cells,and FerroOrange and liperfloo were used to measure the concentration of ferrous ion and the level of lipid peroxidation.A total of 15 female BALB/c-nu mice were randomly divided into control group,DOX group,and DOX+Si-BRD group,with 5 mice in each group,and a subcutaneous inoculation model of MDA-MB-231/DOX cells was established.The methods of qRT-PCR,Western blotting,and immunohistochemistry were used to measure the expression of the BRD4/NF-κB signaling pathway.Results:Compared with the si-NC group,the si-BRD4#1 group and the si-BRD4#2 group had significant reductions in the number of cell clones,positive EDU staining,and glutathione level in MDA-MB-231 and BT549 cells(P<0.001),as well as significant increases in the level of ferrous ion,the level of reactive oxygen species(ROS),and oxidized glutathione/glutathione ratio(P<0.01).Compared with the paren⁃tal cells(MDA-MB-231),there were increases in the mRNA and protein expression levels of BRD4 in MDA-MB-231/DOX cells.Compared with the Con group,the DOX group had significant increases in the expression levels of IKβ-α,NF-κB,and BRD4 and the level of ROS(P<0.05)and significant reductions in the number of cell clones and positive EDU staining(P<0.05);compared with the DOX group,the si-BRD4+DOX group had significant reductions in the expression levels of IKβ-αand NF-κB,the number of cell clones,and positive EDU staining(P<0.05)and a significant increase

关 键 词：溴域蛋白亚家族4 核因子ΚB 铁死亡 乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]