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 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 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 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

Additional file 1: of Role of miR-146a in neural stem cell differentiation and neural lineage determination: relevance for neurodevelopmental disorders

Table S1. Detailed information of all patients and controls whose brain samples were used in this study. Table S2. Possibly deleterious variants in known ASD and ID genes. Table S3. DEGs identified in undifferentiated cells. Table S4. DEGs identified in differentiated cells. Table S5. Validation of RNA-Seq using RT-qPCR on Fluidigm array. Table S6. Top 20 cannonical pathways deregulated in undifferentiated cells. Table S7. Top 4 nodes enriched for protein-protein interaction as calculated by ClusterOne Plugin. Table S8. Top 20 cannonical pathways deregulated in the cell cycle modules of differentiated cells. Table S9. Top 20 cannonical pathways deregulated in the cell neuronal modules of differentiated cells. Table S10. Cell type enrichment analysis of DEGs from the Cell Cycle and Neuronal Modules. (XLSX 888 kb

Additional file 1: of Emergency treatment with levetiracetam or phenytoin in status epilepticus in children—the EcLiPSE study: study protocol for a randomised controlled trial

SPIRIT 2013 checklist: recommended items to address in a clinical trial protocol and related documents. (DOCX 60 kb