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

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（MDA-MB-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1 μmol/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信号通路的激活来抑制乳腺癌化疗诱导的铁死亡。

Abstract

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 1 μmol/L DOX for 24 hours. Colony formation assay and 5-ethynyl-2'-deoxyuridine（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 parental 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-α and NF-κB，the number of cell clones，and positive EDU staining（P<0.05） and a significant increase in the level of ROS（P<0.05）. Compared with the control group，the DOX group had significant reductions in tumor weight and volume（P<0.05） and a significant increase in the number of TUNEL-stained cells in tumor tissue（P<0.05）. In addition，compared with the DOX group，the BRD4+DOX group had further reductions in tumor weight and volume（P<0.001） and a significant increase in the number of TUNEL-stained cells（P<0.001）. Compared with the DOX group，the BRD4+DOX group had significant reductions in Ki-67，BRD-4，and NF-κB（P<0.01） and a significant increase in 4-hydroxynonenal（P<0.01） in tumor tissue. Conclusion BRD4 is an important drug resistance factor，which can inhibit ferroptosis induced by chemotherapy in breast cancer by promoting the activation of the NF-κB signaling pathway.

关键词

溴域蛋白亚家族4 / 核因子κB / 铁死亡 / 乳腺癌

Key words

bromodomain protein subfamily 4 / nuclear factor-kappa B / ferroptosis / breast cancer

基金资助

河北省卫生健康委科研基金资助项目(20190484)