hsa_circ_0006168 drives microglial activation in TNC via miR-99b-5p/KDM6B axis to promote central sensitization in migraine.

BACKGROUND: Migraine is a disabling neurological disorder characterized by recurrent headache attacks and associated symptoms. The mechanisms underlying migraine remain unclear. This study aimed to identify differentially expressed circular RNAs (circRNAs) in migraine and elucidate their potential roles in migraine pathogenesis. METHODS: CircRNA expression was profiled in migraine patients using qRT-PCR. The circular structure and miRNA sponge function of hsa_circ_0006168 (orthologous to mmu_circ_0012878 in mice) were validated by Sanger sequencing, RNase R treatment assay, fluorescence in situ hybridization (FISH), and bioinformatics. Migraine mouse models were established by repeated nitroglycerin (NTG) injections. Lentiviral-mediated knockdown of mmu_circ_0012878, miR-99b-5p agomir, and GSK-J4 (KDM6B inhibitor) were administered to assess their effects on central sensitization, microglial activation, and M1/M2 polarization in the trigeminal nucleus caudalis (TNC) of mice by qRT-PCR, western blot, and immunofluorescence. In vitro, LPS-stimulated BV2 cells with mmu_circ_0012878 knockdown were analyzed for M1/M2 polarization using qRT-PCR, western blot, immunofluorescence, flow cytometry, multiplex flow cytometry assay, and ELISA. Molecular interactions within the mmu_circ_0012878/miR-99b-5p/KDM6B axis were confirmed by qRT-PCR, western blot and dual luciferase assay. RESULTS: hsa_circ_0006168 (mmu_circ_0012878) and KDM6B were upregulated, while miR-99b-5p was downregulated in the peripheral blood of migraine patients and the TNC of NTG-induced migraine mouse models. Silencing mmu_circ_0012878/KDM6B and overexpressing miR-99b-5p attenuated central sensitization, potentially through the PI3K/AKT signaling pathway. Additionally, mmu_circ_0012878 partially colocalized with Ionized calcium-binding adapter molecule 1 (Iba1), and its knockdown suppressed microglial activation and promoted M2 polarization in the TNC. In LPS-stimulated BV-2 cells, mmu_circ_0012878 knockdown reduced microglial activation, shifted polarization toward M2, and decreased M1-related cytokines (IL-6, IL-1β). Dual luciferase assays confirmed mmu_circ_0012878 regulated KDM6B expression by sponging miR-99b-5p. CONCLUSIONS: hsa_circ_0006168 acted as a competitive endogenous RNA (ceRNA) by sponging miR-99b-5p, thereby blocking miR-99b-5p-mediated suppression of KDM6B. The hsa_circ_0006168/miR-99b-5p/KDM6B axis contributed to central sensitization by activating microglia of TNC, possibly via the PI3K/AKT signaling pathway. This study suggested a novel molecular mechanism underlying migraine development and identified potential therapeutic targets for intervention. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s10194-026-02288-0.