mmu-miR-1843b-5p negatively regulates EGR1 expression and modulates GM-CSF-differentiated macrophage activity in experimental autoimmune uveitis

Abstract

To investigate the relationship between mmu-miR-1843b-5p and EGR1 expression during GM-CSF-induced macrophage differentiation in experimental autoimmune uveitis (EAU). Splenic mononuclear cells isolated from EAU mice were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate GM-CSF-differentiated macrophages, whereas cells cultured without GM-CSF served as controls. Gain- and loss-of-function experiments were performed using mmu-miR-1843b-5p mimics, inhibitor, and a non-targeting negative control. Expression levels of mmu-miR-1843b-5p and EGR1 were determined by quantitative PCR, and EGR1 protein expression was assessed by Western blotting. Cell viability was evaluated by CCK-8 assay. Statistical analyses were conducted using analysis of variance and post-hoc tests. During GM-CSF-induced macrophage differentiation, mmu-miR-1843b-5p expression was significantly decreased, whereas EGR1 mRNA and protein levels were increased in GM-CSF-treated cells compared with cells cultured without GM-CSF (all P < 0.01). Transfection with mmu-miR-1843b-5p mimics significantly upregulated mmu-miR-1843b-5p expression and downregulated EGR1 expression, while mmu-miR-1843b-5p inhibition produced the opposite effects (all P < 0.01). Functionally, mmu-miR-1843b-5p overexpression increased the CCK-8 signal in bulk cultures, whereas mmu-miR-1843b-5p inhibition reduced it, with significant differences observed at 48 h and 72 h. Because CCK-8 reflects overall metabolic activity/viability rather than macrophage-specific differentiation, these findings were interpreted cautiously (P < 0.05). These findings indicate a potential role for mmu-miR-1843b-5p in regulating EGR1 expression and GM-CSF-differentiated macrophage activity in EAU. Direct targeting of EGR1 by mmu-miR-1843b-5p and comprehensive M1 functional validation remain to be confirmed.