miR-146a and Astrocyte Inflammatory Response Figure 5. Expression levels of the miR-146a targets (IRAK-1, IRAK-2 and TRAF-6) after transfection with anti-miR-146a LNA or miR- 146a mimic. (A–C) Quantitative real-time PCR of IRAK-1 (A), IRAK-2 (B) and TRAF-6 (C) expression 24 hours after exposure to IL-1b in U373 glioblastoma cell line transfected with LNA-antimiR-146a (25 nM) or miR-146a mimic (pre-mir-146a, 50 nM). (D) Quantitative real-time PCR of IRAK-1, 24 hours after exposure to IL-1b in cultured human astrocytes transfected with LNA anti-miR-146a (50 nM) or miR-146a mimic (pre-mir-146a, 50 nM). Data are expressed relative to the levels in unstimulated cells and are mean 6 SEM from two separate experiments performed in triplicate (E) IRAK-1 protein expression 24 hours after exposure to IL-1b in glial cells transfected with LNA anti-miR-146a (50 nM) or miR-146a mimic (pre-mir-146a, 50 nM); Representative immunoblot (1 control; 2, IL-1b; 3, IL-1b + LNA-antimiR-146a; 4, IL-1b + LNA-antimiR-146a scramble; 5, IL-1b + mimic; 6, IL-1b + mimic scramble) and optical density measurements. Data are expressed relative to the levels in unstimulated cells and are mean 6 SEM from two separate experiments (*p,0.05, compared to control; **p,0.05, LNA or mimic transfected cells stimulated with IL-1b compared to IL-1b alone). doi:10.1371/journal.pone.0044789.g005 dysplastic cortex in FCD IIb. This observation suggests an important role of miR-146a in astrocytes in epilepsy associated lesions. Expression of miR-146a was also detected in balloon cells. Since balloon cells have been shown to represent a major source of proinflammatory molecules and possibly contribute to the TIR signaling [9,41,45], further evaluation of the miR-146a function in these cell types could be performed in recently described culture system [46]. Evaluation of the function of this miRNA in long- term epilepsy associated glioneuronal tumors and in malforma- tions of cortical development (containing balloon cells with a stem cell phenotype), is particularly interesting in view of the recently described function of miR-146a in modulating neural stem cell proliferation and differentiation through regulation of the key neural stem cell factor Notch1 [47]. Further investigation is also needed to evaluate the levels of different miR-146a targets in GG and FCD. miR-146a is Regulated in Human Glial Cells in Culture by IL-1b Since miR-146a was up-regulated in astrocytes in the epilep- togenic lesions (FCD, present results; TLE [20,21]) the expression of miR-146a was further analyzed in culture, using U373 glioblastoma cell line and human fetal astrocytes. We evaluated the expression levels of miR-146a upon to exposure to in- flammatory molecules (IL-1b, IL-6, TNFa or HMGB1) that are known to be up-regulated in epileptogenic tissue from FCD and TLE patients; (for review see [3]) and to the TLR4 ligand LPS. Both cell line and primary astrocytes were found to exhibit a prominent upregulation of miR-146a expression in response to IL-1b stimulation. In contrast, LPS induced modest up-regulation whereas IL-6, TNFa and HMGB1 were not effective in inducing significant changes in miR-146a expression. The effect of IL-1b in human astrocytes is in line with recent studies in culture [39,48], supporting an important role of this cytokine in the regulation of astroglial miR-146a expression. The effect of IL-1b was concen- tration and time dependent and was observed from 16 hours after exposure to the cytokine. The observation that IL-1Ra inhibited the effect of IL-1b on miR-146a is consistent with the fact that IL- 1b signals through the type I IL-1b receptor which is upregulated in epileptogenic lesions [[49]; for review see (Aronica, et al., 2011; Aronica, et al. in press)]. Regulation of IL-1b Pathway by miR-146a To further investigate the function of miR-146a in human glial cells, we studied the effects of overexpression (with miR-146a mimic) or blockade of miR-146a (with antisense miR-146a; LNA- PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44789