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EXPRESSION OF MICRORNAS MIR21, MIR146A, AND MIR155 IN TSC
pression or knockdown experiments targeting all three miRNAs.
To study the effects of miR146a modulation on the IL-1β-mediated inflamma-
tory response we evaluated the levels of IL-6 and COX-2, which have been reported to be associated with astrogliosis in different pathological conditions (for reviews see 81-84) and are prominently induced by IL-1β in human astrocytes, as well as in SEGA cells (33; pres- ent data). The in vitro experiments confirmed in both fetal astrocytes and SEGA cells the negative regulation by miR146a of IL-1β-induced IL-6 mRNA expression. Moreover, COX-2 mRNA levels were decreased by miR146a in unstimulated astrocytes and a ten- dency toward a decreased IL-1β-induced COX-2 mRNA expression was detected in both cell types. The anti-inflammatory role of miR146a is also supported by its ability to regu- late the IL-1β induced release of several other proinflammatory factors, such as HMGB133.
miR155 expression has been shown to be up-regulated in human astrocytes in culture in response to cytokines and TLR ligands 35, 85. In the present study we confirmed the induction of miR155 in response to IL-1β in human fetal astrocytes and in SEGA cells. Similar to miR21, a complex role for this miRNA has been reported in inflammation with both positive and negative regulatory effects on the NF-κB signaling pathway 86; reviewed in 26, 27, although in vitro studies support the positive role in the induction of proinflammatory genes in human astrocytes 85. We show that transfection with miR155 mimic further up-regulated IL-1β-induced IL-6 and COX-2 mRNA levels, in both human fetal astrocytes and SEGA cells, supporting the pro-inflammatory action of this miRNA in astrocytes. Thus, since IL-1β also increased the levels of miR155, its induction may potentially counteract the anti-inflammatory function of miR146. Indeed, cotransfection with miR146a mimic and miR155 inhibitor resulted in additional negative regulation of IL-1β-mediated response. However, the mRNA levels of the miR155 targets, SHIP1 and CFH, which have been suggested to mediate the miR155 proinflammatory effects 79, 87 were not modulated under our experimental conditions and we failed (in contrast to GG 35) to detect a significant negative correlation between miR155 expression and SHIP1 protein expression in tubers. Thus, considering the large number of potential miR155 tar- gets recently detected and validated, further investigation of these target genes will be required to elucidate the specific mechanisms and functions of miR155 on the regulation of the astrocyte-mediated inflammatory response as well as on glial cell proliferation.
A recent study also suggests a key role of this miRNA in inflammation-induced neurogenic deficits via microglial activation and induction of IL-6 88. This is particularly interesting in view of the possible contribution of immunological dysfunctions and microglial activation, occurring early during development, to the pathogenesis of neuro- behavioral disabilities, which are common in TSC patients (for reviews see 89-92). Moreover, miR155 has been shown to play a key role in autophagy targeting multiple players in the mTOR pathway 93. Thus the consequences of miRNA deregulation on both neuronal and glial function via a regulation of the mTOR pathway deserves further investigation.
Inflammation and inflammation-related microRNAs in the astrogliopathology of TSC brain
Our observations point to a deregulation of the inflammatory response in glial cells, which is in line with other studies showing a deregulation of inflammation-related microRNAs in different epilepsy associated pathologies in which gliosis is prominent 24, 25,
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