miR-146a and Astrocyte Inflammatory Response Figure 2. miR-146a expression levels in U373 glioblastoma cell line after exposure to IL-1b. Quantitative real-time PCR of miR-146a expression in U373 cells 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/ml) 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 alone or in the presence of IL-1b). (C) Expression levels of miR-146a 24 hours after exposure to 0.1, 1, 10 or 50 ng/ml of IL-1b. (D) Expression levels of miR-146a in U373 cells incubated for different durations (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.g002 effect was maximal with doses ranging from 1 to 50 ng/ml (Fig. 2 C and Fig. 3 C); increased miR-146a levels were detected 16 hours after exposure to IL-1 ß (Fig. 2 D and Fig. 3 D). Regulation of miR-146a Expression by Transfection with Anti-miR-146a LNA or miR-146a Mimic After transfection with 50 nM LNA-antimiR-146a into U373 glioblastoma cell line (Fig. 4 A) and human fetal astrocytes in culture (not shown) for 24 hours, qPCR revealed significant reduction of miR-146a. On the other hand significant over- expression of miR-146a was seen in U373 glioblastoma cell line (Fig. 4 B) and human fetal astrocytes in culture (not shown) after transfection with miR-146a mimic (pre-miR-146a) for 24 hours at concentrations ranging from 1 to 50 nM. Upon stimulation with 10 ng/ml IL-1ß for 24 hours lower miR-146a levels were observed in cells transfected with LNA-anti miR-146a (but not with the LNA-control) and higher levels in cells transfected with miR-146a mimic (but not by the mimic-control) compared to non-transfected cells (Fig. 4 C and D). Effects of miR-146a Inhibition or Overexpression on its Downstream Targets IRAK-1, IRAK-2 and TRAF-6 Transfection with LNA-anti miR-146a or miR-146a mimic modulated the mRNA expression levels of downstream targets of mir-146a, such as IRAK-1, IRAK-2 and TRAF-6 after exposure to IL-1ß in both U373 (Fig. 5 A–C) and fetal astrocytes (Fig. 5 D, IRAK-1; IRAK2 and TRAF-6: data not shown). In particular, a consistent downregulation of all 3 targets was observed under conditions in which miR-146a was overexpressed. Increased mRNA levels of IRAK-1, IRAK-2 and TRAF-6 were observed after transfection with LNA-anti miR-146a (Fig. 5 B–C, U373; Fig. 5 D, IRAK-1, fetal astrocytes). Western blot analysis showed the downregulation of IRAK-1 protein induced by IL-1ß in both U373 (Fig. 5 E) and fetal astrocytes (not shown). This effect was enhanced by transfection with miR-146a mimic. In contrast LNA- anti miR-146a transfection resulted in the restoration of IRAK1 protein to control level. No significant differences in IRAK-2 and TRAF-6 proteins were observed in either cell culture. PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44789