Xiong Jing, Nie Meifang, Fu Chun, Chai Xiaoshan, Zhang Yongjing, He Ling, Sun Shujuan
Department of Obstetrics and Gynecology, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China.
Stem Cells Int. 2022 Mar 5;2022:1608806. doi: 10.1155/2022/1608806. eCollection 2022.
Cervical cancer (CC) is a prevalent cancer in women. Hypoxia plays a critical role in CC cell ferroptosis resistance. This study explored the mechanism of hypoxia in CC cell ferroptosis resistance by regulating HIF1/KDM4A/H3K9me3.
Cultured SiHa and Hela cells were exposed to CoCl2 and treated with Erastin. Cell viability was detected by MTT assay, and concentrations of iron ion, MDA and GSH were determined using corresponding kits. Expressions of KDM4A, HIF1, TfR1, DMT1, and H3k9me3 were detected by RT-qPCR, Western blot, and ChIP assay. The correlation of KDM4A and HIF1 was analyzed on Oncomine, UALCAN, and Starbase. CC cells were co-transfected with shKDM4A or/and pcDNA3.1-HIF1. Iron uptake and release were assessed using the isotopic tracer method. The binding relationship between HIF1 and HRE sequence was verified by dual-luciferase assay.
Cell viability and GSH were decreased while iron concentration, MDA, KDM4A, and HIF1 levels were increased in hypoxia conditions. The 2-h hypoxia induced ferroptosis resistance. KDM4A and HIF1 were highly-expressed in CC tissues and positively correlated with each other. KDM4A knockdown attenuated cell resistance to Erastin, increased H3K9me3 level in the HIF1 promoter region, and downregulated HIF1 transcription and translation. H3K9me3 level was increased in the HIF1 promoter after hypoxia. HIF1 overexpression abrogated the function of KDM4A knockdown on ferroptosis in hypoxia conditions. Iron uptake/release and TfR1/DMT1 levels were increased after hypoxia. Hypoxia activated HRE sequence in TfR1 and DMT1 promoters.
Hypoxia upregulated KDM4A, enhanced HIF1 transcription, and activated HRE sequence in TfR1 and DMT1 promoters via H3K9me3, thus inducing ferroptosis resistance in CC cells.
宫颈癌（CC）是女性中一种常见的癌症。缺氧在CC细胞铁死亡抗性中起关键作用。本研究通过调节HIF1/KDM4A/H3K9me3探索缺氧在CC细胞铁死亡抗性中的机制。
将培养的SiHa和Hela细胞暴露于CoCl2并使用艾拉司群处理。通过MTT法检测细胞活力，使用相应试剂盒测定铁离子、丙二醛（MDA）和谷胱甘肽（GSH）的浓度。通过RT-qPCR、蛋白质免疫印迹法和染色质免疫沉淀（ChIP）试验检测KDM4A、HIF1、转铁蛋白受体1（TfR1）、二价金属离子转运体1（DMT1）和H3k9me3的表达。在Oncomine、UALCAN和Starbase上分析KDM4A和HIF1的相关性。将CC细胞与shKDM4A或/和pcDNA3.1-HIF1共转染。使用同位素示踪法评估铁的摄取和释放。通过双荧光素酶试验验证HIF1与缺氧反应元件（HRE）序列之间的结合关系。
在缺氧条件下，细胞活力和GSH降低，而铁浓度、MDA、KDM4A和HIF1水平升高。2小时的缺氧诱导铁死亡抗性。KDM4A和HIF1在CC组织中高表达且彼此呈正相关。敲低KDM4A可减弱细胞对艾拉司群的抗性，增加HIF1启动子区域的H3K9me3水平，并下调HIF1的转录和翻译。缺氧后HIF1启动子中的H3K9me3水平升高。HIF1过表达消除了缺氧条件下敲低KDM4A对铁死亡的作用。缺氧后铁摄取/释放以及TfR1/DMT1水平升高。缺氧激活了TfR1和DMT1启动子中的HRE序列。
缺氧通过H3K9me3上调KDM4A，增强HIF1转录，并激活TfR1和DMT1启动子中的HRE序列，从而诱导CC细胞产生铁死亡抗性。
