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which is in agreement with previous studies 34, 35, 47-49. Expression of miR155 was also detected in endothelial cells. In line with this, miR155 expression in blood vessels has been reported in GG 35 and this miRNA has been shown to be implicated in endothelial cell functions and angiogenesis 50-53.
The positive correlation observed between miR21, miR146a and miR155 and the expression of GFAP in tubers supports the role of astrocytes as source and targets of regulation of these miRNAs. Accordingly, miR21, miR146a 34 and miR155 (unpublished observations) are prominently upregulated in the hippocampus after induction of status epilepticus (SE) during the latent period preceding the onset of seizures which is asso- ciated with prominent astrogliosis and increased levels of pro-inflammatory cytokines, such as IL-1β 54, 55. Up-regulation of miRNA146a in astrocytes has also been detected in hippocampal specimens of patients with temporal lobe epilepsy (TLE) in regions with astrogliosis 54, 56. In addition, we recently reported expression of miRNA155 in gangliogli- oma (GG) with increased expression in peritumoral tissue (also in reactive astrocytes) compared with the tumor 35.
Strong expression of these miRNAs was also detected in SEGAs, which are low- grade, slow-growing glial tumors that usually develop in children and adolescents with TSC and represent a major cause of morbidity and mortality 57, 58. miR21 recently emerged as one of the important dysregulated miRNAs in gliomas, possibly involved in the regu- lation of migration and survival of glioma cells 59-62. Increasing evidence supports the key role of miR155 in both inflammation and oncogenesis (for review see 26, 63-65), as well as in inflammation-induced oncogenesis 66. miR146a has also been recently suggested to modulate glial cells proliferation and differentiation 67. Thus, further studies are needed to determine whether the expression levels of these miRNA would influence SEGA cells proliferation and differentiation, as well as tumor recurrence.
Regulation of astrocyte-mediated inflammatory response
Our in vitro findings support the role of IL-1β as major inducer of these miRNAs in both human astrocytes and SEGA cells 33, 35; present data). Moreover a positive correlation was observed between these miRNAs and IL-1β in both tumor and peritumoral cortex in GG 35 and in TSC specimens (miR21 and miR155, present data).
Previous studies have shown that miR21 can be induced by NF-κB and may act as negative-feedback regulator of Toll-like receptor signaling via targeting of the pro-in- flammatory tumor suppressor PDCD4 27, 28, 61, 68, 69. In the present study we confirmed the intracellular induction of this microRNA by IL-1β in both astrocytes and SEGA cells. This observation is also supported by the positive correlation detected in cortical tubers between miR21 levels and the expression of IL-1β. Several studies indicate a complex role for miR21 in the modulation of inflammation with both positive and negative regulatory effects 64 The anti-inflammatory effects of miR21 have been attributed to the targeting of PCDC4 which results in an increase of the anti-inflammatory cytokine interleukin-10 64, 70. However, in our study, although transfection with miR21 mimic decreased the mRNA expression level of PDCD4 in both astrocytes and SEGA cells, the increased level of this microRNA did not interfere with IL-1β-mediated induction of IL-6 or COX-2. Moreover, we did not detect any significant correlation between miR21 expression in cortical tubers and its targets PDCD4, PTEN, Neurotrophin-3 and MEF2C.