Fig. 3.
Natural compound similars regulate a set of fibrosis-associated miRNAs. a Treatment with compounds led to upregulation of several miRNAs across all treatments, whereas downregulation of a set of miRNAs was observed in buf-s-14, buf-s-18, lycorine as well as lyco-s. miRNAs analysed in more detail indicated on the right. n = 3 biological replicates (donors), 2 technical replicates each. Discoveries determined with multiple unpaired t tests, adjusted following BKY (Q = 0.01). Pathways of significantly deregulated miRNAs, summarized by “bufalin-like” (blue) or “lycorine-like” (red) treatment, were determined by over-representation analysis (ORA) using miEAA2, adjusted following BH (Q = 0.05). A high number of terms were found related to extracellular matrix (b), fibrosis and fibroblast biology (c) as well as TGF-β pathways (d). As the analysis included more similars of bufalin than lycorine, more significant terms were observed for “bufalin-like” treatment overall. Dotted line indicates significance threshold (padj. < 0.05). Real-time qPCR analysis showed hsa-miR-125a-5p expression was independent of compound treatment (e), whereas hsa-miR-132-3p was upregulated in compound-treated HCF (f). n = 1 donor, 3 biological replicates. Analysed with Two-way ANOVA, adjusted following Tukey. Utilizing the WST-1 assay, overexpression of hsa-miR-125a-5p partially protected against the anti-proliferative effect of buf-s-14, buf-s-18 and lyco-s (g). In contrast, inhibition of hsa-miR-132-3p protected against buf-s-18 and lyco-s, but not buf-s-14 (h). Bars show Lipofectamine control (hatched), overexpression with respective miRNA mimic (full colour) and inhibition with respective miRNA inhibitor (pale colour). Horizontal lines represent remaining activity after treatment with buf-s-14 (pink, dash-dot-dot) and buf-s-18 (teal, dash-dot) without miRNA mimic or inhibitor. n = 3 biological replicates (donors), 3–10 technical replicates each. Analysed with Two-way ANOVA (p < 0.05), adjusted following Tukey