FIGURE 4.
miR‐146a‐5p increased NOTCH signalling through interaction with and downregulation of NUMB expression. (a) Heatmap of significantly differentially expressed mRNA (FDR < 0.05) from NHA cells treated with a miR‐146a‐5p mimic for 48 h and compared to a scrambled control (miR‐NC). (b) Significantly down‐regulated mRNA results were compared to three databases (TargetScan 7.1, miRBD and microT‐CDS) and 18 common genes were found as in situ projected binding partners. (c) mRNA‐pulldown assay followed by qPCR quantification of bound mRNA‐miR‐146a‐5p complexes from the common 18 binding partners. (d) qPCR of NUMB expression in NHA cells after co‐culture with 5.0 × 109 EVs from melanocytes, NHA or MBM cell lines (H1, H2 and H3), normalized with U6 expression. (e) WB of Numb protein expression in NHA cells after co‐culture with 5.0 × 109 EVs from melanocytes, NHA or MBM cell lines (H1, H2 and H3). (f) qPCR of NUMB expression in NHA cells after co‐culture with miR‐146a‐5p mimic or a scrambled control, normalized to U6 expression. (g) Western blot of Numb protein expression in NHA cells after co‐culture with miR‐146a‐5p mimic or a scrambled control. (h) Sequence of miR‐146a‐5p 3p binding site, and the comparison of NUMB 3′UTR‐wild type (WT) and mutated (MUT) region. (i) Dual luciferase assay on the WT or MUT 3′UTR region with miR‐146a‐5p or a scrambled control in NHA cells, represented as the relative firefly to Renilla luciferase activity. (j) ELISA of top four upregulated cytokines released by NHA cells with WT or overexpression of NUMB in the presence of miR‐146a‐5p mimic or control. n.s.= not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001.