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MIR147B: A NOVEL KEY REGULATOR OF IL-1β-MEDIATED INFLAMMATION IN ASTROCYTES
has not been described in the epileptogenic brain. mRNA-sequencing identified sev- eral GO terms enriched after IL-1β stimulation of fetal astrocytes, related to immune response and inflammatory signaling. Correspondingly, the pathway enrichment analysis showed up-regulation of specific pathways related to inflammation like the NF-κB and TNF signaling pathway, confirming the activation of the targeted pathways by IL-1β stim- ulation. By inhibiting the NF-κB pathway in these cultures using TPCA-1, an inhibitor of both the IκB kinase-2 (IKK-2), which plays a crucial role NF-κB-regulated production of pro-inflammatory molecules 31, and STAT3, which is also implicated in regulation of IL-6 and COX-2 transcripts 32, we confirmed the specific involvement of this pathway in the up-regulation of miR146a and miR147b after stimulation with IL-1β. Finally, mRNA-seq analysis also showed a strong positive correlation between the expression of miR147b and the expression of C15orf48, which is also known as NMES1 27. The pre-miRNA of miR147b is transcribed from a region within the 3’ UTR of C15orf48, and since little is known about C15orf48, its strong up-regulation, co-expression and relation to miR147b may warrant further investigation for its role in inflammation.
miR147b mimic acts both under inflammatory and basal conditions on a dis- integrin and metalloproteinase15 (ADAM15), which contributes to blood-brain barrier dysfunction and inflammation by increasing vascular permeability and leukocyte migra- tion 33. It was previously shown that ADAM15 was targeted by miR147b in human vascular endothelial cells, hereby attenuating albumin passage across endothelial monolayers in vitro 26. Thus, the upregulation of miR147b under inflammatory conditions may serve as protective mechanism restoring blood-brain barrier dysfunction. This may be highly beneficial in the epileptogenic brain, as blood-brain barrier dysfunction may contribute to progression of epilepsy 34.
miR147b overexpression decreased the level of DEPTOR, a mTOR-interacting protein 35, which is activated in both human and experimental epileptogenic brain 36-38. Interestingly, DEPTOR is involved in negative regulation of the mTOR pathway, by inhib- iting mTORC1 signaling 35. In the same study, reduction of DEPTOR was also associated with apoptosis and activation of PI3K signaling leading to increased proliferation, how- ever, under our experimental conditions, we did not observe any of these effects. Recent studies suggest also direct inhibitory effects of miR147b on Akt and mTOR activation 28, 29 that deservers further investigation.
miR147b reduces pro-inflammatory mediators
We found that artificial overexpression of miR147b during inflammatory conditions in fetal astrocyte and TSC cell cultures led to decreased expression of pro-inflammatory cytokines IL-6 and COX-2 and complement component 3 (C3). IL-6 and COX-2 have been reported to be associated with astrogliosis in different pathological conditions 39 and are highly up-regulated in astrocytes after stimulation with IL-1β 19. C3 was recently indicated as one of the most characteristic and highly up-regulated genes in human A1 astrocytes, which are defined as harmful reactive astrocytes that up-regulate classical complement cascade genes and are often found in brain regions associated with disease 40. Downregulation of C3 might indicate either a decrease in reactivity of the astrocytes or a shift towards the more protective A2 type astrocyte 40. Thus, miR147b seems to act like a negative regulator of IL-1β induced inflammation, similar to what has been
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