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.

<p>(<b>A–C</b>) Quantitative real-time PCR of IRAK-1 (A), IRAK-2 (B) and TRAF-6 (C) expression 24 hours after exposure to IL-1β in U373 glioblastoma cell line transfected with LNA-antimiR-146a (25 nM) or miR-146a mimic (pre-mir-146a, 50 nM). (<b>D</b>) Quantitative real-time PCR of IRAK-1, 24 hours after exposure to IL-1β 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 ± SEM from two separate experiments performed in triplicate (<b>E</b>) IRAK-1 protein expression 24 hours after exposure to IL-1β 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-1β; 3, IL-1β + LNA-antimiR-146a; 4, IL-1β + LNA-antimiR-146a scramble; 5, IL-1β + mimic; 6, IL-1β + mimic scramble) and optical density measurements. Data are expressed relative to the levels in unstimulated cells and are mean ± SEM from two separate experiments (*p<0.05, compared to control; **p<0.05, LNA or mimic transfected cells stimulated with IL-1β compared to IL-1β alone).</p

Effect of anti-miR-146a LNA or miR-146a mimic upon IL-1β-induced release of inflammatory molecules.

<p>(<b>A</b>) Cytokine release 24 hours after exposure to IL-1β 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 ± 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-γ, IP-10, MIP-1β, and TNF-α (* 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.</p

Effect of anti-miR-146a LNA or miR-146a mimic upon IL-1β-induced IL-6 and COX-2 mRNA.

<p>Quantitative real-time PCR of IL-6 (A–B) and Cox-2 (C–D). (<b>A–B</b>) IL-6 mRNA levels, 24 hours after exposure to IL-1β 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). (<b>C–D</b>) COX-2 mRNA levels, 24 hours after exposure to IL-1β 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 ± 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-1β compared to IL-1β alone).</p

miR-146a expression in GG and FCD type IIb.

<p>(<b>A</b>) Quantitative real-time PCR of miR-146a in control cortex (n = 6; autopsy), FCD type IIb (n = 6) and GG (n = 5) specimens. miR-146a expression was normalized to that of the U6B small nuclear RNA gene (rnu6b). The error bars represent SEM; statistical significance: *P<0.05 compared to control. (<b>B–E</b>) In situ hybridization of miR-146a expression in control (B–C) and FCD type IIb (D–E) specimens. miR-146a was expressed at low levels in neurons and undetectable in glial cells in control grey (B) and white matter (C) specimens. Panels D: FCD type IIb tissue showing miR-146a expression in glial cells (arrows; insert a); inserts (b,c) in D show colocalization (purple) of miR-146a (red) in GFAP (blue) positive astrocytes. Panel E: FCD type IIb tissue showing miR-146a expression in balloon cells (arrows). Scale bar in B: B: 80 µm. C–E: 40 µm.</p

Effect of anti-miR-146a LNA or miR-146a mimic upon IL-1β-induced COX-2 protein and release of HMGB1.

<p>COX-2 protein expression 24 hours after exposure to IL-1β in U373 cells transfected with LNA-antimiR-146a (50 nM) or miR-146a mimic (pre-mir-146a, 50 nM). (<b>A</b>) Representative immunoblot and (<b>B</b>) densitometric analysis: values (optical density units relative to the optical density of β-actin) are mean ± SEM of two separate experiments performed and are expressed relative to the levels in unstimulated cells. (<b>C</b>) HMGB1 immunoblot (1 control; 2, IL-1β; 3, IL-1β + mimic; 4, IL-1β + mimic scramble; 5 mimic; 6, IL-1β + LNA-antimiR-146a; 7, IL-1β + LNA-antimiR-146a scramble 8, LNA) and densitometric analysis (<b>D</b>, optical density units of cellular HMGB1 relative to the optical density of β-actin). *p<0.05, compared to control; **p<0.05, LNA or mimic transfected cells stimulated with IL-1β compared to IL-1β alone.</p

miR-146a expression levels after transfection with anti-miR-146a LNA or miR-146a mimic (pre-miR146a).

<p>Quantitative real-time PCR of miR-146a. (<b>A</b>) miR-146a expression after transfection with LNA-antimiR-146a (50 nM) in U373 cells; insert shows in green tranfected cells (<b>B</b>) miR-146a expression after transfection of miR-146a mimic (pre-mir-146a, 1, 25 and 50 nM). (<b>C</b>) miR-146a expression 24 hours after exposure to IL-1β in U373 cells transfected with LNA-antimiR-146a (50 nM) or miR-146a mimic (pre-mir-146a, 50 nM). (D) miR-146a expression 24 hours after exposure to IL-1β in cultured human astrocytes 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 ± 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-1β, compared to IL-1β alone).</p

miR-146a expression levels in cultured human astrocytes after exposure to IL-1β.

<p>Quantitative real-time PCR of miR-146a expression in human fetal astrocytes in culture. (<b>A</b>) Expression levels of miR-146a 24 hours after exposure to IL-1β (10 ng/ml) or LPS (100 ng/ml) in the presence or absence of the IL-1β receptor antagonist (IL-1ra; 1 µg/ml) or LPS-RS (10 µg/m) respectively. (<b>B</b>) Expression levels of miR-146a 24 hours after exposure to IL-1β (10 ng/ml), TNFα (1 ng/ml), IL-6 (10 ng/ml), HMGB1 (40 nM). (<b>C</b>) Expression levels of miR-146a 24 hours after to 0.1, 1, 10 or 50 ng/ml of IL-1β. (<b>D</b>) 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-1β (10 ng/ml). Data are expressed relative to the levels observed in unstimulated cells and are mean ± SEM from two separate experiments performed in triplicate (*p<0.05 compared to control).</p

miR-146a expression levels in U373 glioblastoma cell line after exposure to IL-1β.

<p>Quantitative real-time PCR of miR-146a expression in U373 cells in culture. (<b>A</b>) Expression levels of miR-146a 24 hours after exposure to IL-1β (10 ng/ml) or LPS (100 ng/ml) in the presence or absence of the IL-1β receptor antagonist (IL-1ra; 1 µg/ml) or LPS-RS (10 µg/ml) respectively. (<b>B</b>) Expression levels of miR-146a 24 hours after exposure to IL-1β (10 ng/ml), TNFα (1 ng/ml), IL-6 (10 ng/ml), HMGB1 (40 nM alone or in the presence of IL-1β). (<b>C</b>) Expression levels of miR-146a 24 hours after exposure to 0.1, 1, 10 or 50 ng/ml of IL-1β. (<b>D</b>) 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-1β (10 ng/ml). Data are expressed relative to the levels observed in unstimulated cells and are mean ± SEM from two separate experiments performed in triplicate (*p<0.05 compared to control).</p