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MIR147B: A NOVEL KEY REGULATOR OF IL-1β-MEDIATED INFLAMMATION IN ASTROCYTES
examined expression levels of the pro-neurogenesis signaling components brain-derived neurotrophic factor, paired box protein Pax-6 and sex determining region Y-box 4, which were predicted to be regulated by miR147b. However, the expression of these genes was not altered (data not shown).
miR147b expression in tuberous sclerosis complex cortical tubers and temporal lobe epilepsy with hippocampal sclerosis
Expression and localization of miR147b in TSC and TLE-HS brain tissue was examined with qPCR of fresh brain tissue and ISH (Fig 6 A-H), respectively. miR147b expression was increased 3.5-fold in TLE-HS (p<0.0001, Supp. Fig 2), and in a subset of TSC tuber homog- enates (n=4 out of 16, ranging from 2.1-4.2-fold increase, Supp. Fig 2). ISH showed neu- ronal expression of miR147b in control cortex and hippocampus. In TSC cortex miR147b expression was localized in dysmorphic neurons, giant cells and astrocytes in the tuber (co-localization with GFAP is depicted in inset in D), and in perituberal neurons. miR147b expression was evident in TSC white matter astrocytes and giant cells whereas in control white matter miR147b was not detected. In TLE-HS hippocampus, increased expression of miR147b was observed in remaining neurons and reactive astrocytes (co-localization with GFAP is depicted in inset in H) within sclerotic areas with gliosis.
miR147b and inflammatory mediators in tuberous sclerosis complex cortical tuber cell cultures
To validate the findings from fetal astrocytes, TSC cell cultures were transfected with miR147b mimic. miR147b overexpression during IL-1β stimulation led to reduced IL-6 and COX-2 mRNA expression levels by approximately 31 and 28%, respectively (p=0.0022 and p<0.0043 respectively, Fig 6 I-J). There was a trend towards reduced C3 expression after transfection with miR147b mimic (p=0.0931, Fig 6K).
Discussion
We aimed to identify which miRNAs are produced by fetal human astrocytes during inflammatory conditions in vitro, in order to find miRNAs that could be a therapeutic target. We identified miR146a and miR147b to be differentially expressed in fetal astro- cytes after IL-1β stimulation, which was associated with increased expression of genes related to immune response and inflammation. Overexpression of miR147b reduced the expression of the pro-inflammatory mediators IL-6 and COX-2 after IL-1β stimulation. Transfection of cell cultures with miR146a and miR147b mimics decreased proliferation of fetal astrocytes and promoted neuronal differentiation of NSCs. ISH showed increased expression of miR147b in astrocytes in resected brain tissue from patients with TLE-HS and TSC, as compared to controls.
Increased expression of miR146a and miR147b in fetal astrocytes after IL-1β stimulation
Sequencing analysis identified miR146a and miR147b as the two main miRNAs involved in IL-1β mediated inflammation in fetal astrocytes. miR146a was previously described in relation to epilepsy and inflammation in astrocytes 16-19. miR147b, however, has been described in endothelial cells 26, macrophages 27 and different types of cancer 28-30, but
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