Fig. 7

Dysregulation of H19X-encoded miRNAs in APs affects their differentiation into beige adipocytes. A, B Cells were cultured in a differentiation medium for beige fat lineage and harvested for analysis at several time points (n = 3). A Mature miRNAs, miR-322-5p and miR-542-3p, were downregulated in differentiated beige adipocytes. B The primary transcripts of miR-503 and miR-542 were downregulated immediately upon the differentiation of adipocytes. The let-7b primary transcript was used as a control. C-E APs were transiently transfected with miRNA mimic mix (miR-322-5p/miR-503-5p/miR-450b-5p/miR-542-3p) for 24 h and stimulated with an adipogenic cocktail for the differentiation. Cells were harvested for analysis at several time points after the induction of differentiation (day 0, 2, 4, 6, and 8) (C) The representative images of Oil Red O staining in differentiated beige adipocytes at day 8 (left panel). The quantified intensities of Oil Red O staining are shown (right panel). The pretreatment of miRNA mimic mix reduced the number of differentiated adipocytes. Scale bar, 100 µm (D) The mRNA levels of Ucp1 and Ap2 were analyzed during beige adipocyte differentiation (n = 2). The pretreatment of miRNA mimic mix (miR-322-5p/miR-503-5p/miR-450b-5p/miR-542-3p) attenuated the expression of Ucp1 and Ap2. E The protein levels of CCND1 and PLIN1 were examined by western blotting during beige adipocyte differentiation. miRNA mimic mix downregulated CCND1 protein levels and delayed the expression of PLIN1 protein. Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05