Yan Dawei, Wan Lei
The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui, China.
Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of IHM, Hefei, China.
Front Immunol. 2025 Jun 10;16:1601259. doi: 10.3389/fimmu.2025.1601259. eCollection 2025.
Interactions between fibroblast-like synoviocytes (FLSs) and macrophages are pivotal in the pathogenesis of rheumatoid arthritis (RA). Exosomal circular RNAs (circRNAs) are key players in the communication between RA-FLSs and macrophages, yet their specific roles in RA pathogenesis remain undefined.This study aims to investigate the specific regulatory mechanisms of exosomal circRNAs in RA and their function in macrophage polarization through in vitro experiments.
RA-FLSs were stimulated with TNF-α to mimic the enhanced inflammatory microenvironment in RA, while exosome secretion was inhibited using GW4869. WTAP expression was modulated via transfection (overexpression or knockdown), and m⁶A modification levels were analyzed using MeRIP-qPCR. Protein-RNA interactions, macrophage polarization, and cytokine profiles were evaluated through RNA pull-down assays, RIP-qPCR, flow cytometry, and ELISA, respectively.
In the enhanced inflammatory microenvironment of RA, circ-CBLB expression was observed to be significantly downregulated. Further functional validation showed that inhibition of exosome secretion intensified macrophage polarization toward the pro-inflammatory M1 phenotype. Screening of mA modification-related enzymes combined with RNA pull-down and RIP-qPCR assays exhibited that WTAP protein directly bound to circ-CBLB and accelerated circ-CBLB degradation by enhancing its mA modification levels. Functional experiments demonstrated that WTAP overexpression decreased exosomal circ-CBLB levels and promoted macrophage polarization toward the pro-inflammatory M1 phenotype, which was reversed by mA modification site mutation.
This study reveals a novel mechanism that exosomal circ-CBLB secreted from RA-FLSs affects macrophage polarization under the regulation of WTAP-mediated mA modification, underscoring the potential of interventions targeting the WTAP-circ-CBLB axis in RA treatment.
成纤维样滑膜细胞（FLS）与巨噬细胞之间的相互作用在类风湿关节炎（RA）的发病机制中起关键作用。外泌体环状RNA（circRNA）是RA-FLS与巨噬细胞之间通讯的关键参与者，但其在RA发病机制中的具体作用仍不明确。本研究旨在通过体外实验探究外泌体circRNA在RA中的具体调控机制及其在巨噬细胞极化中的作用。
用肿瘤坏死因子-α（TNF-α）刺激RA-FLS以模拟RA中增强的炎症微环境，同时使用GW4869抑制外泌体分泌。通过转染（过表达或敲低）调节WTAP表达，并使用甲基化RNA免疫沉淀定量PCR（MeRIP-qPCR）分析m⁶A修饰水平。分别通过RNA下拉试验、RNA免疫沉淀定量PCR（RIP-qPCR）、流式细胞术和酶联免疫吸附测定（ELISA）评估蛋白质-RNA相互作用、巨噬细胞极化和细胞因子谱。
在RA增强的炎症微环境中，观察到circ-CBLB表达显著下调。进一步的功能验证表明，抑制外泌体分泌会加剧巨噬细胞向促炎M1表型的极化。筛选与m⁶A修饰相关的酶并结合RNA下拉和RIP-qPCR试验表明，WTAP蛋白直接与circ-CBLB结合，并通过提高其m⁶A修饰水平加速circ-CBLB降解。功能实验表明，WTAP过表达降低了外泌体circ-CBLB水平，并促进巨噬细胞向促炎M1表型极化，而m⁶A修饰位点突变可逆转这一现象。
本研究揭示了一种新机制，即RA-FLS分泌的外泌体circ-CBLB在WTAP介导的m⁶A修饰调控下影响巨噬细胞极化，强调了针对WTAP-circ-CBLB轴进行干预在RA治疗中的潜力。
