miR-146a and Astrocyte Inflammatory Response Figure 3. miR-146a expression levels in cultured human astrocytes after exposure to IL-1b. Quantitative real-time PCR of miR-146a expression in human fetal astrocytes in culture. (A) Expression levels of miR-146a 24 hours after exposure to IL-1b (10 ng/ml) or LPS (100 ng/ml) in the presence or absence of the IL-1b receptor antagonist (IL-1ra; 1 mg/ml) or LPS-RS (10 mg/m) respectively. (B) Expression levels of miR-146a 24 hours after exposure to IL-1b (10 ng/ml), TNFa (1 ng/ml), IL-6 (10 ng/ml), HMGB1 (40 nM). (C) Expression levels of miR-146a 24 hours after to 0.1, 1, 10 or 50 ng/ml of IL-1b. (D) Expression levels of miR-146a in cells incubated for different times (10, 30, 60 min and 6, 16, 24, 48) hours in the presence of IL-1b (10 ng/ml). Data are expressed relative to the levels observed in unstimulated cells and are mean 6 SEM from two separate experiments performed in triplicate (*p,0.05 compared to control). doi:10.1371/journal.pone.0044789.g003 Effects of miR-146a Inhibition or Overexpression on IL-1b Mediated Induction of IL-6 and COX-2 IL-1ß induced expression of two major inflammatory mediators, IL-6 and Cox-2 was modulated by transfection of glial cells with LNA-anti miR-146a or miR-146a mimic. Transfection with miR- 146a mimic downregulated IL-1ß induced IL-6 and Cox- 2 mRNA levels in both U373 glioblastoma cells and human fetal astrocytes in culture (Fig. 6 A–D). In contrast, LNA anti-miR-146a transfection significantly increased the levels of IL-6 and Cox-2 induced by IL-1ß (Fig. 6 A–D). Western blot analysis confirmed the different effects of LNA- anti-miR-146a and miR-146a mimic on COX-2 protein expres- sion in IL-1ß stimulated cells; transfection with miR-146a mimic reduced whereas LNA-anti-miR-146a increased the COX-2 expression (Fig. 7 A–B). Effects of miR-146a Inhibition or Overexpression on IL-1b Mediated Release of Inflammatory Mediators We evaluated the release of high mobility group box (HMGB1; by western blot analysis) and the release of MCP1 (by ELISA) in response to IL-1ß stimulation in both U373 and fetal astrocytes. Transfection of U373 with miR-146a mimic decreased both the cellular and extracellular levels of HMGB1 (Fig. 7 C–D) compared to IL-1ß stimulated cells. The release of MCP-1 induced by IL-1ß was negatively regulated by transfection with miR-146a mimic (MCP-1 pg/ml: control: 15826193; IL-1ß: 2715161368; IL-1ß + miR-146a mimic: 1105462027; p,0.05 compared to IL-1ß stimulated cells), whereas transfection with LNA-anti miR-146a did not significantly affect the release induced by IL-1ß. The release of 16 cytokines/chemokines was evaluated using the human magnetic 10-plex panel in U373 glioblastoma cell line. IL- 5, IL-17 and IFN-a2 were under detection limits in our culture conditions. The amount of IL-10, IL-13, IL-18 and VEGF did not significantly change after the different treatments. IL-1b signifi- cantly increased the release IL-6, IL-8, IFN-c, IP-10, MIP-1b, G- CSF, Rantes and TNF-a (Fig. 8). Transfection with LNA-anti miR-146a before treatment with IL-1b, significantly increased the levels of IL-6 and IP-10; whereas transfection with miR-146a mimic before treatment with IL-1b significantly decreased the PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44789