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EXPRESSION OF MICRORNAS MIR21, MIR146A, AND MIR155 IN TSC
cells, inhibition of miR21 with anti-miR21 LNA increased IL-6 expression under stimulated conditions (Fig. 7G; P=0.0087) and a similar trend can be seen for COX-2 in fetal and SEGA (Fig. 7D,J). In fetal astrocytes a modest decrease of IL-6 and increase of COX-2 was observed after miR21 mimic transfection under basal conditions (Fig. 7A,D; P=0.0011 and P=0.0001, respectively).
Transfection with miR146a mimic down-regulated IL-1β-induced IL-6 mRNA lev- els in both human fetal astrocytes and SEGA cells (Fig. 7B,H; P=0.0089 and P=0.0043, respectively); decreased expression of IL-6 was also detected under basal conditions in fetal astrocytes (Fig. 7B; P<0.0001). In contrast, anti-miR146a LNA transfection sig- nificantly increased the levels of IL-6 induced by IL-1β in SEGA cells (Fig. 7H; P=0.0411). COX-2 mRNA levels were decreased by miR146a mimic under basal condition in fetal astrocytes (Fig. 7E; P=0.0015) and a tendency toward a decreased expression was also detected under stimulated conditions in both cell types (Fig. 7E,K). In contrast, anti- miR146a LNA transfection significantly increased the levels of COX-2 under basal con- ditions in both fetal and SEGA cells (Fig. 7E,K; P<0.0001 and P=0.0022, respectively). Further, inhibition of miR146a increased the levels of COX-2 induced by IL-1β in SEGA cells (Fig. 7K; P=0.0411), and a similar trend can be seen in fetal astrocytes (Fig. 7E).
Transfection with miR155 mimic up-regulated IL-1β-induced IL-6 and COX-2 mRNA levels, in both human fetal astrocytes and SEGA cells (Fig. 7C,F,I,L; IL-6: P=0.0355, COX-2: P=0.0015 in fetal astrocytes and IL-6: P=0.026, COX-2: P=0.026 in SEGA-derived cells). Increased expression of IL-6 and COX-2 was also detected under basal conditions (P<0.0001 for both in fetal astrocytes and P=0.0022 for both in SEGA-derived cells). Anti- miR155 LNA transfection increased the levels of COX-2 mRNA only under basal condi- tions in fetal astrocytes (Fig. 7F; P<0.0001), but not in the other experimental conditions. Co-transfection with miR146a mimic and anti-miR155 LNA resulted in additional decrease of IL-1β-induced IL-6 and COX-2 mRNA levels fetal cells (P=0.0339 and P<0.0001, respec- tively), and on COX-2 mRNA levels in SEGA cultures (P=0.0106) compared with miR146a mimic transfection alone (Supp. Fig. 3).
We also evaluated the release of IL-6 in response to IL-1β stimulation by ELISA. Transfection with the miR146a mimic (but not miR21 or miR155) reduced the release induced by IL-1β in fetal astrocytes (P=0.0294; data not shown).
Discussion
This study provides supportive evidence of the induction of inflammation-related miR- NAs associated with the activation of the IL-1R/TLR pathway in TSC brain lesions, high- lighting the differential role of these microRNAs in the regulation of the glia mediated inflammatory response. The cell-specific distribution of miR21, miR146a, and miR155 in TSC brain lesions (tuber and SEGA), as well as the effects of their modulation upon IL-1β-mediated glial inflammatory and possible contribution to astrogliopathology in TSC, are discussed in the following paragraphs.
Inflammation-related microRNAs in TSC brain lesions
In the present study we observed in a large cohort of TSC specimens, increased expres- sion of three inflammation-related miRNAs, miR21, miR146a and miR155. ISH showed expression in dysmorphic neurons, giant cells and reactive astrocytes within the tuber,
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