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MIR147B: A NOVEL KEY REGULATOR OF IL-1β-MEDIATED INFLAMMATION IN ASTROCYTES
pal (male/female: 9/5; years/range: 25-86). None of these patients had a history of seizures or other neurological diseases. Tissue was obtained and used in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee. The local ethical committees of all participating centers gave permission to undertake the study.
In situ hybridization
In situ hybridization (ISH) for miR147b was performed on 5 μm thick tissue sections using a double digoxygenin (DIG)-labeled probe (with LNA modification every third nucleo- tide, Exiqon, Vedbaek, Denmark) as described previously 17.
Statistical analysis
Statistical analysis of cell culture experiments was performed with GraphPad Prism® software (Graphpad software Inc., La Jolla, CA, USA) using the non-parametric Mann- Whitney U test or, for multiple groups, the non-parametric Kruskall-Wallis test with correction for multiple comparisons (Dunn’s method). Correlations were assessed with SPSS (IBM Corp., Armonk, NY, USA) using the Spearman’s rank correlation test. P<0.05 was assumed to indicate a significant difference.
Results
Small RNA-Sequencing
To explore changes in small non-coding RNA expression in astrocytes after IL-1β- stimulation, small RNA-Seq was carried out on control and IL-1β stimulated fetal astro- cyte cultures. Each sequencing run produced ~11 million paired-end reads per sample. After quality assessment and filtering, ~7.5 million paired-end reads remained for each sample, of which ~65% were mapped to the genomic locations of various small RNA species. The expression of 881 small RNAs species across the control and IL-1β stimu- lated cultures was detected, this included 518 miRNAs and 295 small nucleolar RNAs. Of these 881 small RNAs, two miRNAs, miR147b and miR146a, were identified as differentially expressed and up-regulated 3.78-fold (adjusted p-value<0.033) and 5.35-fold (adjusted p-value<0.0008), respectively, in IL-1β stimulated cultures (Fig 1A).
mRNA-Sequencing
mRNA-Seq was carried out on the same set of samples that underwent small RNA- Seq. Each sample was sequenced to the depth of ~21 million paired-end reads. Overall ~16 million passed quality assessment and filtering, of which ~84% were concordantly mapped to the human reference genome GRCh38. Overall, there were 79 differen- tially expressed genes (DEGs); 71 genes were up-regulated in IL-1β stimulated cultures (adjusted p-value<0.05), while 8 were down-regulated after IL-1β stimulation (adjusted p-value<0.05, Fig 1B, Supp. Table 2).
A GO enrichment analysis of the 79 DEGs revealed 16 significantly enriched GO terms (adjusted p-value<0.05) across the categories biological process, cellular com- partment and molecular function (Fig 1C, Table 1). The subsequent pathway enrichment analysis identified enriched pathways related to immune response and inflammation, including cytokine-cytokine receptor interaction and numerous viral infection pathways
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