miR-146a and Astrocyte Inflammatory Response Figure 6. Effect of anti-miR-146a LNA or miR-146a mimic upon IL-1b-induced IL-6 and COX-2 mRNA. Quantitative real-time PCR of IL-6 (A–B) and Cox-2 (C–D). (A–B) IL-6 mRNA levels, 24 hours after exposure to IL-1b in U373 cells (A) and cultured human astrocytes (B) transfected with LNA-antimiR-146a (50 nM) or miR-146a mimic (pre-mir-146a, 50 nM). (C–D) COX-2 mRNA levels, 24 hours after exposure to IL-1b in U373 cells (C) and cultured human astrocytes (D) transfected with LNA-antimiR-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 (*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.g006 anti-miR-146a) on the expression levels of downstream signaling molecules (IRAK-1, IRAK-2 and TRAF-6) of the TIR signaling pathway that have previously been shown to be miR-146 targets [15,17–19]. In both the glioblastoma cell line and human astrocytes transfection with miR-146a mimic, significantly reduced IRAK-1, IRAK-2 and TRAF-6 mRNA and IRAK-1 protein after stimulation with IL-1b. In a previous study in primary cultures of human astroglial cells exposed to IL-1b and Ab42 peptide, it has been shown that an antisense miRNA-146a inhibits miRNA-146a and controls IRAK-1, but not IRAK-2 expression, suggesting an independent regulation of the two targets under these experimen- tal conditions [19]. However, in our study, both IRAK-1 and IRAK-2 mRNA were positively regulated by LNA-anti-miR-146a (and negatively by miR-146a mimic) in IL-1b stimulated human astrocytes. This observation argues against a proinflammatory role of this miRNA-146a, as result of a compensatory upregulation of IRAK2 (at least in the absence of Ab42 peptide). However, the results at the protein level may suggest a preferential regulation of IRAK-1, which is prominently downregulated by miR-146a mimic. The effect of miR-146a on multiple targets may also vary in different cell types and upon different stimuli. For example, it has been shown that miR-146a is upregulated during viral infection in macrophages and acts as a negative regulator of the retinoic acid-inducible gene I (RIG-I)-like helicases by targeting not only IRAK1 but also TRAF6 and IRAK2 [50]. miR-146a Regulates the Expression IL-6 and COX-2 To further evaluate the function of miR-146a in response to inflammatory stimuli, we investigated the effect of miR-146a overexpression or reduction on IL-6 and COX-2, two major inflammatory molecules induced by IL-1b [for reviews see [51,52]]. Astrocytes are a major source of IL-6 and prominent induction of this cytokine on IL-1b stimulation has been previously reported in human fetal astrocytes in culture [53,54]. Both IL-6 and COX-2 have been reported to be associated with astrogliosis and activation of the innate immune response in different pathological conditions (for reviews see [3,51,52]). Furthermore, miR-146a has a complementarity with the COX-2 39 UTR and miR-146a mimic has been recently shown to reduce COX- 2 mRNA levels in fibroblasts [55] and glial cells [56]. The in vitro overexpression and knock-down studies showed that transfection with miR-146a mimic reduces the IL-6 and COX- 2 mRNA levels in cultures stimulated with IL-1b, whereas LNA- anti-miR-146a had the opposite effect. Western blot analysis PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44789