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CODING AND SMALL NON-CODING TRANSCRIPTIONAL LANDSCAPE OF TSC
expression (log2 transformed) of significantly differential genes between TSC and con- trol subjects revealed the innate immune response and extracellular matrix organization modules harbored the majority of significantly elevated genes in TSC patients (Fig. 3b and c). Genes with significantly decreased expression in TSC patients were predomi- nantly located in modules attuned to neuronal functions, for example the neurogen- esis and glutamate receptor signaling module (Fig. 3b and c). Next, we sought to infer miRNA that potentially target genes within the gene co-expression network. To this aim we firstly leveraged the module eigengene concept (module first principal component) to test for correlations between miRNA expression indices and module expression pat- terns. Here, we focused our attention on miRNA with elevated patterns of expression in TSC patients relative to controls. In particular, we identified significant indirect negative correlations between the glutamate receptor signaling and neurogenesis modules with expression indices of miR193, miR200, and the miR34 families (Pearson’s correlation) (Supplementary Table 3). Using the miRWalk 2.0 database51 we identified multiple genes in the neurogenesis and glutamate receptor signaling modules that are predicted targets of miR34a, miR34b and/or miR34c (Fig. 3d). No overlap was found between the putative miR34 family targets in the modules neurogenesis and glutamate receptor signaling and any cell-specific genes. Altogether, these results suggest that neurogenesis and gluta- mate receptor signaling modules may be targeted and, in turn, regulated by specific miRNA families altered in TSC brain tissue.
Cellular distribution of selected over-expressed miRNAs in TSC cortical tubers and control cortex
Thus far our findings suggest that typical neuronal cellular pathways were predicted targets of specific miRNA family members. Considering our analysis was centered upon complex brain tissue, which encompasses multiple cell-types, we here sought to gain insight into the cell-specific patterns of selected miRNA expression in TSC and control brain tissue, that is, the miR34 family members, miR34a and miR34b. In line with our RNA- seq data, in situ hybridization targeting miR34a-5p and miR34b-5p showed low expression in control cortex for both miRNAs (Fig. 4a,e) and high expression in TSC tubers, specif- ically dysmorphic neurons, giant cells and in cells with astroglial morphology (Fig. 4b,d; Fig. 4f,h). Double labeling confirmed the expression of these miRNAs in NeuN (neurons) – and GFAP (astrocytes)- positive cells in TSC cortical tuber specimens (Fig. 4).
Overexpression of miR34a-5p increases expression of IL-1β in human fetal astrocytes
To study the impact of miR34a-5p and miR34b-5p overexpression in fetal astrocytes two different assays were performed. First, in an attempt to induce miR34a-5p and miR34-5p expression, fetal astrocytes were stimulated with IL-1β. No increase in the expression levels of miR34a-5p and miR34b-5p due to IL-1β were observed. Subsequently, the fetal astrocytes were transfected with a miR34a-5p and a miR34b-5p mimic. Fetal astro- cytes transfected with the miR34b-5p mimic had an associated up-regulation of IL-1β (~4-Fold, p-value<0.03) and the inflammatory marker IL-6 (~2.3-fold, p-value<0.03, data not shown). Conversely, no increase in IL-1β and IL-6 levels was seen after transfection with the miR34a-5p mimic, and a down regulation of COX-2 (~1.5-fold, p-value<0.03) was
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