Fig. 9: RNAseq reveals downregulation of multiple factors in critical OL differentiation and myelination pathways with overexpression of miR-145.

a Fluorescence micrographs of primary differentiating OLs transfected with scrambled control (miR-ctl; left panel) or miR-145 mimic (miR-145; right panel) on differentiation day 5. Cells are stained for F-actin (red), Olig2 (green) and MBP (magenta) and counterstained with Hoechst. b Principal component analysis (PCA) of all mapped transcripts for all samples. Plot shows two-dimensional comparison of N = 2 for miR-ctl and miR-145 calculated using the DESeq2 plotPCA function and rlog-transformed count data. c Heat map displaying clustering analysis for N = 2 for miR-ctl and miR-145. d Volcano plot of log2 fold change versus log10 adjusted p value for all mapped transcripts after filtering for miR-145 relative to miR-ctl. Fold change was calculated using DESeq2, p value was adjusted using the Benjamini-Hochberg method. e Significantly enriched gene ontology (GO) terms from genes differentially downregulated in miR-145 OLs relative to miR-ctl, sorted by overarching category. f All differentially downregulated genes identified by GO term analysis overarching categories. g Validation of selected significantly differentially expressed genes by qRT-PCR. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test adjusted for multiple propagations. Analysed by ΔΔCt, expressed as log2 fold change relative to miR-ctl. Genes in bold/red bars indicate confirmed/putative targets of miR-145-5p.