Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4

Abstract

Perturbed expression of microRNAs (miRs) has been reported in different diseases includingautoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR25-3p and its targets in the central nervous system (CNS) tissue from mice with experimentalautoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets inactivated macrophages and splenocytes.EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyteglycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We nextestablished primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and antiCD3/anti-CD28 antibodies, respectively.MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosisfactor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression ofphosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak ofthe disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significantupregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show anyalterations in miR-25-3p expression compared with vehicle-treated cells.Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators duringautoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.