Liu Jialin, Pan Ke, Yang Chunli, Jiang Mengjie, Cheng Jiangling, He Yao, Wang Aichun, Gou Hongman
Department of Clinical Laboratory, Mianyang Maternity and Child Healthcare Hospital & Mianyang Children's Hospital, Mianyang, Sichuan, 621000, China.
Reprod Sci. 2025 Aug 13. doi: 10.1007/s43032-025-01938-z.
Preeclampsia (PE) is a severe and prevalent complication of pregnancy, characterized by insufficient trophoblast invasion, placental hypoxia, and systemic inflammation, which collectively represent the central pathological mechanisms underlying the disease. The molecular mechanism involving activating transcription factor 2 (ATF2), ubiquitin-specific peptidase 4 (USP4), and lysine methyltransferase 2 A (KMT2A) in hypoxia/reoxygenation (H/R)-treated trophoblast cells (HTR-8/SVneo) is explored, aiming to provide a novel theoretical basis for therapeutic strategies against PE. An in vitro H/R model was established using HTR-8/SVneo trophoblast cells to simulate PE pathophysiology. The expression levels of mRNA and protein were evaluated using RT-qPCR and western blotting, respectively. The proliferation, apoptosis, and invasion of cells were assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays. Ferroptosis-related markers were measured using commercial kits. The interaction between USP4 and KMT2A was determined by co-immunoprecipitation (Co-IP) and ubiquitination assays. Dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays were employed to investigate the binding of ATF2 to the USP4 promoter. KMT2A knockdown enhanced hypoxia/reoxygenation (H/R)-induced proliferation and invasiveness and suppressed apoptosis and ferroptosis in HTR-8/SVneo cells. The deubiquitinase USP4 stabilized KMT2A protein expression through deubiquitination. Overexpression of KMT2A reversed the impacts of USP4 silencing on H/R-mediated cellular injury and ferroptosis. Mechanistically, ATF2 transcriptionally activated USP4 expression. Furthermore, USP4 upregulation rescued the influences of ATF2 depletion on H/R-induced HTR-8/SVneo cell injury and ferroptosis. ATF2 activated USP4 transcription, which subsequently deubiquitinated and stabilized KMT2A protein expression, thereby influencing the progression of PE.
子痫前期（PE）是一种严重且常见的妊娠并发症，其特征为滋养层细胞浸润不足、胎盘缺氧和全身炎症，这些共同构成了该疾病的核心病理机制。本研究探讨了缺氧/复氧（H/R）处理的滋养层细胞（HTR-8/SVneo）中涉及激活转录因子2（ATF2）、泛素特异性蛋白酶4（USP4）和赖氨酸甲基转移酶2A（KMT2A）的分子机制，旨在为子痫前期的治疗策略提供新的理论依据。使用HTR-8/SVneo滋养层细胞建立体外H/R模型以模拟子痫前期的病理生理学。分别使用RT-qPCR和蛋白质印迹法评估mRNA和蛋白质的表达水平。使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）、5-乙炔基-2'-脱氧尿苷（EdU）、流式细胞术和Transwell实验评估细胞的增殖、凋亡和侵袭。使用商业试剂盒检测铁死亡相关标志物。通过免疫共沉淀（Co-IP）和泛素化实验确定USP4与KMT2A之间的相互作用。采用双荧光素酶报告基因和染色质免疫沉淀（ChIP）实验研究ATF2与USP4启动子的结合。KMT2A基因敲低增强了缺氧/复氧（H/R）诱导的HTR-8/SVneo细胞增殖和侵袭能力，并抑制了细胞凋亡和铁死亡。去泛素酶USP4通过去泛素化作用稳定KMT2A蛋白表达。KMT2A过表达逆转了USP4沉默对H/R介导的细胞损伤和铁死亡的影响。机制上，ATF2转录激活USP4表达。此外，USP4上调挽救了ATF2缺失对H/R诱导的HTR-8/SVneo细胞损伤和铁死亡的影响。ATF2激活USP4转录，随后USP4使KMT2A蛋白去泛素化并使其稳定，从而影响子痫前期的进展。
