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EXPRESSION OF MICRORNAS MIR21, MIR146A, AND MIR155 IN TSC
tuber and SEGA specimens was investigated using ISH (Figs. 2-4). In both control and perituberal cortex, miR21 was expressed at low levels in neuronal cells (Fig. 2A-C); only occasionally expression was detected in glial cells in perituberal cortex (Fig. 2C). miR21 expression was more evident within the tuber; we detected miR21 expression in cells with astroglial morphology, in dysmorphic neurons and in giant cells (Fig. 2D-I). Double labeling confirmed miR21 expression in NeuN-, GFAP-, and pS6-positive cells, whereas no detectable expression was observed in HLA-DR positive cells, indicating microglia and macrophages (Fig. 2F-I). miR21 was encountered in all the SEGA specimens examined, with moderate to strong expression in GFAP- and pS6-positive cells (Fig. 2J-M). In con- trol cortex miR146a was expressed at low levels in neuronal cells, but was undetectable in glial cells (Fig. 3A,B). miR146a expression was increased in the tuber (Fig. 3D-F) compared with control and perituberal cortex (Fig. 3C). We detected miR146a expression in cells with typical astroglial morphology, in dysmorphic neurons and giant cells (Fig. 3E-G); double labeling confirmed miR146a expression in NeuN-, GFAP-, and pS6-positive cells, whereas no detectable expression was observed in HLA-DR positive cells (not shown). miR146a was encountered in all the SEGA specimens examined, with expression in GFAP- and pS6-positive cells (Fig. 3H). Expression of miR155 in control and perituberal cortex was mainly observed in neuronal cells, but was undetectable in glial cells (Fig 4A-C). In tubers miR155 was expressed in both dysmorphic neurons, glial and giant cells (NeuN- and GFAP- positive cells; Fig. 4D-H); we also observed expression of miR155 in blood vessels (Fig. 4E-F; co-localization with CD34). Expression of miR155 was also encountered in SEGA specimens (Fig. 4I; GFAP- and pS6-positive cells, not shown).
miRNA target expression in TSC
Evaluation of mRNA expression levels of downstream targets of miR21 (PTEN, PDCD4, Neurotrophin-3 and MEF2C), miR146a (IRAK1, IRAK2 and TRAF6, ERBB4 and NUMB), and miR155 (SHIP1), showed up-regulation of IRAK2 and TRAF6 in TSC compared with controls (data not shown; pairwise comparison following Kruskal-Wallis, IRAK1: P<0.05, IRAK2: P<0.05, TRAF6: P<0.05, NUMB: P<0.05); only a tendency toward an increase was detected for SHIP1 (P<0.057). Expression of miR146a correlated with the downstream target IRAK1 (r=0.4770, P=0.0077). Since these target genes are known to be expressed in different cell types, we evaluated the expression and cellular distribution of PDCD4, TRAF6, IRAK1 and SHIP1 in tubers by immunohistochemistry. As previously reported 35 expression in glial cells was observed for all targets evaluated. However, we detected a negative correlation only between the TRAF6 and IRAK1 IRS and miR146a expression (TRAF6: r=-0.427, P=0.0, IRAK1: r=-0.377, P=0.03).
Regulation of miR21, miR146a and miR155 expression by IL-1β in cell culture
In the present study we used both human fetal astrocytes and SEGA-derived cells in cul- ture to examine the effect of IL-1β on miR21, miR146a and miR155 expression. qPCR analy- sis demonstrated that exposure to IL-1β increased miR21, miR146a and miR155 expression in both astrocytes and SEGA cells (Fig. 5A,C, miR21: P=0.0042, miR146a: P<0.0001, miR155: P<0.0001 in fetal astrocytes and miR21: P=0.0226, miR146a: P<0.0001, miR155: P=0.0011 in SEGA-derived cells). The induction of miR146a was prominent and was associated with extracellular release in both cell cultures (Fig. 5B,D; P=0.0008 in fetal and P=0.0012 in
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