Dong Min, Xu Min, Fang Derong, Chen Yiyuan, Zhang Mingzhe
Reproductive Medicine Center, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, China.
Reproductive Medicine Center, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2025 Jul;41(7):603-610.
Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.
目的 探讨泛素特异性蛋白酶21（USP21）增加叉头框蛋白M1（FOXM1）稳定性并促进子宫内膜异位症（EM）中巨噬细胞M2极化的机制。方法 培养从患者收集的在位子宫内膜基质细胞（EESC）和常规健康体检者的正常子宫内膜基质细胞（NESC），采用RT-qPCR和蛋白质免疫印迹法检测USP21和FOXM1的表达水平。将EESC与巨噬细胞共培养，采用RT-qPCR检测白细胞介素6（IL-6）和CXC趋化因子配体10（CXCL10）的M1极化标志物以及CD206和纤连蛋白1（FN1）的M2极化标志物。通过流式细胞术进一步检测M2标志物CD206。采用酶联免疫吸附测定（ELISA）检测细胞上清液中IL-6、肿瘤坏死因子-α（TNF-α）、IL-10和转化生长因子-β（TGF-β）水平。采用免疫共沉淀法评估USP21与FOXM1之间的相互作用以及FOXM1的泛素化水平。通过放线菌酮（CHX）试验检测FOXM1蛋白稳定性。结果 与NESC组相比，EESC组中USP21和FOXM1表达水平显著升高；与NESC + M0组相比，EESC + M0组中M1极化标志物（IL-6和CXCL10）的表达无显著差异，但M2极化标志物（CD206和FN1）的表达增加，同时M2巨噬细胞数量显著增加；细胞上清液中IL-6和TNF-α水平无显著差异，但IL-10和TGF-β水平升高。上述结果表明去泛素化酶USP21在EM中高表达，促进巨噬细胞M2极化。敲低USP21或FOXM1可抑制EM巨噬细胞的M2极化。USP21在EESC中与FOXM1相互作用，导致FOXM1泛素化水平降低，FOXM1蛋白稳定性增加。FOXM1过表达逆转了敲低USP21对EM巨噬细胞M2极化的抑制作用。结论 去泛素化酶USP21与FOXM1相互作用，增加FOXM1稳定性并促进EM巨噬细胞M2极化。
