Fig. 6

The negative feedback loop of miRNA/Wnt affects the proliferation and beige commitment in IL4-treated Aps. A LiCl treatment reduced the abundance of mature miRNAs to less than half that observed in control APs. miR-322-5p, miR-351-5p, and miR-542-3p were examined by qPCR as representatives of H19X-encoded miRNAs (n = 3). B The mRNA expression levels of Wnt signaling–related genes were measured by qPCR (n = 3). LiCl treatment increased the mRNA levels of Ccnd1 and Fzd6. The transfection of miRNA mimics together with LiCl treatment alleviated the effect of the Wnt activator, LiCl. C, D The proliferation of APs was analyzed by MTT assay (n = 3). C LiCl treatment enhanced the cell proliferation, and transfection of miR-322-5p/miR-503-5p and miR-450b-5p/miR-542-3p mimics lowered the LiCl-induced proliferation. D IL-4-induced cell proliferation was suppressed by the transfection of miR-322-5p/miR-503-5p and miR-450b-5p/miR-542-3p mimics. E, F The proliferation of APs was analyzed by flow cytometry (E) and immunocytochemistry (F). The percentage of Ki67-positive cells increased after six days of incubation in IL-4 containing medium but decreased upon additional transfection of miR-322-5p/miR-503-5p mimics. Scale: 100 µm G The mRNA expression levels of beige adipogenic markers were measured by qPCR (n = 3). IL-4-induced upregulation of Pparg and Pgc1-α expressions was compromised by LiCl treatment. Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05.