CCAAT/enhancer-binding protein delta regulates miRs-4257 and 3156 to attenuate the interleukin 12 through small extracellular vesicle transmission in glioblastoma

Glioblastoma multiforme (GBM) is a heterogeneous and malignant brain tumor characterized by an immunosuppressive microenvironment, notably with diminished M1 macrophage activity. MicroRNAs serve as post-transcriptional regulators and have been implicated in influencing tumor progression. However, the interaction between microRNAs and tumor-associated macrophages in glioma remains less characterized. CCAAT/enhancer-binding protein delta (CEBPD) could act as an oncogenic factor in GBM and regulate microRNA transcription, thereby impacting tumorigenesis. In this study, we demonstrated that glioma CEBPD directly binds to and activates the promoter regions of miR-4257 and miR-3156, located within the genes ADAMTSL4 and ANKRD30BP3, respectively. These microRNAs are transmitted via small extracellular vesicles (sEVs) and target macrophages, specifically binding to the 3'-untranslated regions (UTRs) of interleukin 12 (IL-12) p35 and p40 mRNAs, thereby reducing IL-12 transcription and expression in macrophages. Furthermore, our results show that sEV antisense miR-4257 and miR-3156 diminish the M1 macrophage phenotype. In animal models, co-inoculation of glioma cells with antisense miR-4257 and miR-3156 or CEBPD knockdown, along with M1 macrophages, leads to reduced tumor growth and enhanced M1 macrophage activation. These findings suggest that glioma CEBPD can contribute to immunosuppression by regulating miR-4257 and miR-3156, which target IL-12 in macrophages through sEV transmission. This research offers new insights into the relationship between glioma and immunosuppression, highlighting potential therapeutic avenues for enhancing anti-tumor immunity in GBM.