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Figure 5 Effects of IL-1β stimulation on miR21, miR146a and miR155 expression in cultures. Quan- titative real-time PCR of miRNA expression intracellular and release in the supernatant fraction in human fetal astrocytes and SEGA-derived cell cultures. A and C. Expression levels of miR21, miR146a and miR155 after 24 hours exposure to IL-1β (10 ng/ml). Stimulation with IL-1β increased expression of all three microRNAs compared to control. B and D. Extracellular release of miR21, miR146a and miR155 after 24 hours exposure to IL-1β. Stimulation with IL-1β increased miR146a re- lease in the medium. Data are expressed relative to the levels observed in untreated cells and are mean ± SEM from five (A) or three (B, C and D) experiments on cultures derived from separate donors performed in triplicate. *p<0.05, **p<0.01, ***p<0.001 compared to control, Mann Whitney U test. SEGA: subependymal giant cell astrocytomas.
IRAK2 mRNA expression were observed after transfection of both miR146a mimic and anti-miR146a LNA compared with control and IL-1β stimulated fetal astrocytes (Supp. Fig. 2D). However in SEGA-derived cells anti-miR146a LNA positively modulated the levels of IRAK2 mRNA upon IL-1β stimulation (Supp. Fig. 2H; P=0.0152).
SHIP1 mRNA expression was not modulated by transfection with miR155 mimic or anti-miR155 LNA under our experimental conditions (not shown). No changes of complement factor H (CFH) mRNA expression were observed after transfection of all three miRNA mimics.
Effects of miR21, miR146a and miR155 inhibition or overexpression on IL-1β- mediated induction of IL-6 and COX-2 mRNA
We also evaluated the effect of modulation of miRNA levels in culture on IL-1β- induced expression of two major inflammatory mediators, IL-6 and cyclo-oxygenase-2 (COX-2) (Fig. 7A-L).
Transfection with miR21 mimic or anti-miR21 LNA did not affect the levels of IL-6 and COX-2 under stimulated conditions in fetal cells (Fig. 7A,D). In SEGA-derived