miR-146a and Astrocyte Inflammatory Response Figure 7. Effect of anti-miR-146a LNA or miR-146a mimic upon IL-1b-induced COX-2 protein and release of HMGB1. COX-2 protein expression 24 hours after exposure to IL-1b in U373 cells transfected with LNA-antimiR-146a (50 nM) or miR-146a mimic (pre-mir-146a, 50 nM). (A) Representative immunoblot and (B) densitometric analysis: values (optical density units relative to the optical density of b-actin) are mean 6 SEM of two separate experiments performed and are expressed relative to the levels in unstimulated cells. (C) HMGB1 immunoblot (1 control; 2, IL-1b; 3, IL- 1b + mimic; 4, IL-1b + mimic scramble; 5 mimic; 6, IL-1b + LNA-antimiR-146a; 7, IL-1b + LNA-antimiR-146a scramble 8, LNA) and densitometric analysis (D, optical density units of cellular HMGB1 relative to the optical density of b-actin). *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.g007 Figure 8. Effect of anti-miR-146a LNA or miR-146a mimic upon IL-1b-induced release of inflammatory molecules. (A) Cytokine release 24 hours after exposure to IL-1b in U373 cells transfected with LNA-antimiR-146a (50 nM) or miR-146a mimic (miR-146a mimic, 50 nM). Data are expressed relative to unstimulated control cells (mean 6 SEM from three separate experiments). In comparison with IL-1ß alone, cultures stimulated with IL-1ß and transfected with LNA-anti miR-146a exhibited significant increase of IL-6 and IP-10 release, whereas transfection of glial cells with miR- 146a mimic significantly decreased the levels of IL-6, IL-8, G-CSF, IFN-c, IP-10, MIP-1b, and TNF-a (* p,0.05). LNA-anti miR-146a and miR-146a mimic alone did not significantly affect the levels of cytokines in the culture medium, compared to non treated cells. doi:10.1371/journal.pone.0044789.g008 PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e44789