Xiong Jing, Chen Puxiang, He Ling, Chai Xiaoshan, Zhang Yongjing, Sun Shujuan
Department of Obstetrics and Gynecology, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China.
Environ Toxicol. 2024 Aug;39(8):4207-4220. doi: 10.1002/tox.24304. Epub 2024 May 10.
The discovery of ferroptosis has unveiled new perspectives for cervical cancer (CC) management. We elucidated the functional mechanism of hypoxia-like conditions in CC cell ferroptosis resistance. CC cells were subjected to normoxia or hypoxia-like conditions, followed by erastin treatment to induce ferroptosis. The assessment of cell viability/ferroptosis resistance was performed by MTT assay/Fe, MDA, and glutathione measurement by colorimetry. KDM4A/SUMO1/Ubc9/SENP1 protein levels were determined by Western blot. Interaction and binding sites between KDM4A and SUMO1 were analyzed and predicted by immunofluorescence/co-immunoprecipitation and GPS-SUMO 1.0 software, with the target relationship verified by mutation experiment. SLC7A11/GPX4/H3K9me3 protein levels, and H3K9me3 level in the SLC7A11 gene promoter region were determined by RT-qPCR and Western blot/chromatin immunoprecipitation. H3H9me3/SLC7A11/GPX4 level alterations, and ferroptosis resistance after KDM4A silencing or KDM4A K471 mutation were assessed. Hypoxia-like conditions increased CC cell ferroptosis resistance and KDM4A, SUMO1, and Ubc9 protein levels, while it decreased SENP1 protein level. KDM4A and SUMO1 were co-localized in the nucleus, and hypoxia-like conditions promoted their interaction. Specifically, the K471 locus of KDM4A was the main locus for SUMO1ylation. Hypoxia-like conditions up-regulated SLC7A11 and GPX4 expression levels and decreased H3K9me3 protein level and H3K9me3 abundance in the SLC7A11 promoter region. KDM4A silencing or K471 locus mutation resulted in weakened interaction between KDM4A and SUMO1, elevated H3K9me3 levels, decreased SLC7A11 expression, ultimately, a reduced CC cell ferroptosis resistance. CoCl-stimulated hypoxia-like conditions enhanced SUMO1 modification of KDM4A at the K471 locus specifically, repressed H3K9me3 levels, and up-regulated SLC7A11/GPX4 to enhance CC cell ferroptosis resistance.
铁死亡的发现为宫颈癌（CC）的治疗开辟了新的视角。我们阐明了缺氧样条件在CC细胞铁死亡抗性中的功能机制。将CC细胞置于常氧或缺氧样条件下，然后用erastin处理以诱导铁死亡。通过MTT法评估细胞活力/铁死亡抗性，通过比色法测量铁、丙二醛和谷胱甘肽。通过蛋白质印迹法测定KDM4A/SUMO1/Ubc9/SENP1蛋白水平。通过免疫荧光/免疫共沉淀和GPS-SUMO 1.0软件分析和预测KDM4A与SUMO1之间的相互作用和结合位点，并通过突变实验验证靶标关系。通过RT-qPCR以及蛋白质印迹法/染色质免疫沉淀法测定SLC7A11/GPX4/H3K9me3蛋白水平以及SLC7A11基因启动子区域的H3K9me3水平。评估KDM4A沉默或KDM4A K471突变后H3H9me3/SLC7A11/GPX4水平变化以及铁死亡抗性。缺氧样条件增加了CC细胞的铁死亡抗性以及KDM4A、SUMO1和Ubc9蛋白水平，同时降低了SENP1蛋白水平。KDM4A和SUMO1共定位于细胞核，缺氧样条件促进了它们的相互作用。具体而言，KDM4A的K471位点是SUMO化的主要位点。缺氧样条件上调了SLC7A11和GPX4的表达水平，降低了SLC7A11启动子区域的H3K9me3蛋白水平和H3K9me3丰度。KDM4A沉默或K471位点突变导致KDM4A与SUMO1之间的相互作用减弱，H3K9me3水平升高，SLC7A11表达降低，最终降低了CC细胞的铁死亡抗性。CoCl刺激的缺氧样条件特异性增强了K471位点处KDM4A的SUMO1修饰，抑制了H3K9me3水平，并上调了SLC7A11/GPX4以增强CC细胞的铁死亡抗性。
