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SEGA-derived cells).
Regulation of miR21, miR146a and miR155 expression and their targets by trans- fection with miRNA inhibitor or miRNA mimic
Fetal astrocytes and SEGA-derived cells in culture were used to examine the effect of specific anti-miRNA LNA or miRNA mimic oligonucleotides upon IL-1β induction of miR21, miR146a and miR155 (Fig. 6A-F). qPCR revealed that transfection with 50 nM anti-miRNA LNA for 24 h could significantly and consistently reduce the IL-1β-induced up-regulation of the corresponding miRNA in almost all cases in both cell types (miR21: P<0.0001, miR146a: P<0.0001, miR155: P<0.0001 in fetal astrocytes and miR21: P<0.0001, miR146a: P=0.0073, miR155: P>0.05 in SEGA-derived cells). In contrast, under basal con- ditions, only miR21 expression was reduced after transfection with anti-miRNA LNA (Fig. 6A,D; P<0.001 for both cell types) and miR146 levels were slightly increased compared with control in fetal astrocytes (Fig. 6B; P<0.001) but not in SEGA cells (Fig. 6E).
After transfection with miRNA mimic, overexpression of the specific miRNA was seen in fetal astrocytes and SEGA-derived cells under both basal and stimulated con- ditions (Fig. 6A-F; P<0.001 for all three miRNAs in both cell types). Although miR21 has been shown to regulate miR155 expression by promoting the expression of IL-10, acting as negative regulator of miR155 expression 26, 27, the levels of miR155 were not influenced by miR21 transfection under our experimental conditions. LNA negative control and mimic negative control did not affect the levels of miRNA expression. Cell viability was not influenced by anti-miRNA LNA or miRNA mimic transfections and IL-1β treatment (data not shown).
Since the transfection of small RNAs may result in complex effects on gene regulation 45, 46 we evaluated the effect of miRNA overexpression or knockdown on the expression of specific mRNA targets.
RT-PCR analysis was performed to evaluate the effect of modulation in cul- ture of miR21 and miR146a levels on the mRNA expression of putative targets related to inflammatory pathways, such as the IL-1R/TLR. Transfection with miR21 mimic signifi- cantly decreased the mRNA expression level of PDCD4 under both basal and stimulated conditions (Supp. Fig. 2A,E; P<0.0001 for both basal and stimulated conditions in fetal astrocytes, P=0.0022 under basal and P=0.0152 under stimulated conditions in SEGA- derived cells), whereas the effect of anti-miR21 LNA on PDCD4 was variable, showing no changes under stimulated conditions and even a slight decrease of PDCD4 under basal conditions (Supp. Fig. 2A, E; P=0.0232 for fetal astrocytes, P=0.0152 for SEGA-derived cells).
Transfection with miR146a mimic decreased the mRNA expression level of TRAF6 (Supp. Fig. 2B,F; P=0.0007 for fetal astrocytes, P=0.0022 for SEGA-derived cells) and IRAK1 (Supp. Fig. 2C,G; P<0.0001 for fetal astrocytes, P=0.0022 for SEGA-derived cells), whereas transfection with anti-miR146a LNA increased the expression of these targets under both basal and stimulated conditions in fetal astrocytes (Supp. Fig. 2B-C; TRAF6: P<0.0001 under basal, P=0.0002 under stimulated conditions; IRAK1: P<0.0001 under basal, P=0.0007 under stimulated conditions). In SEGA-derived cells, anti-miR146a LNA increased the expression of TRAF6 under stimulated conditions (Supp. Fig. 2F; P=0.0152), under basal conditions only a trend was visible (P=0.0649). No significant changes of