Figures & data
Figure 1. Inverse regulation of miR125b-5p and MMP11 during human adipogenesis. (A) Representative phase contrast images of cells at 0-12 d after adipogenic induction. (B) PPARG, (C) FABP4, (D) ATGL, (E) MMP11 (white bars), miR125b-5p (black bars) and miR125b-3p (gray bars) mRNA levels and (F) MMP11 protein levels during adipogenesis. MMP11 protein levels were normalized to total protein amount in each sample. The data are presented as mean+-SEM of these 2 independent adipocyte differentiation courses. (G) Homology comparison of predicted miR125b-5p binding sites in the 3′UTR region of human MMP11. Conserved miR125b-5p binding sites between different species are indicated by a bold sequence. For standardization, target gene expression was normalized to the mean of the 2 housekeeping genes: β-actin (ACTB), and TATAbox-binding protein (TBP). *, p < 0.05; ***, p < 0.001, ns, not significant.
Figure 2. MiR125b-5p targets MMP11 by binding to the 3′UTR. (A) MiR125b-5p overexpression (pre-miR125b-5p) and downregulation (anti-miR125b-5p) efficiency was verified by qRT-PCR and normalized to scrambled control (pre-ntRNA, anti-ntRNA). (B) MMP11 mRNA and protein levels after overexpression or downregulation of miR125b-5p. (C) MMP11 mRNA expression after stratification in tertiles according to miR125b-5p mRNA levels in isolated human adipocytes of children. (D) Luciferase reporter plasmids carrying wild-type (WT) or mutant (MUT) miR125b-5p binding sites (gray boxes). MUT(1-3) indicate individual mutations of the single binding site; MUT(1.2.3), mutation of all binding sites. The insertion of the mutation is indicated in bold. (E) Relative luciferase activity after co-transfection of control or MMP11 with pre-miR125b-5p/pre-ntRNA or antimiR125b-5p/anti-ntRNA in SGBS preadipocytes. In addition, relative luciferase activity after cotransfection of control or mutated versions of miR125b-5p binding sites with pre-miR125b-5p/pre-ntRNA or anti-miR125b-5p/anti-ntRNA in SGBS preadipocytes. For standardization, target gene expression was normalized to the mean of the 2 housekeeping genes: β-actin (ACTB), and TATAbox-binding protein (TBP). *, p < 0.05; ***, p < 0.001.
Figure 3. MMP11 knockdown only mildly affected adipogenesis. (A) SiRNA-mediated MMP11 knockdown efficiency was verified at mRNA level on day 0, 12 of adipogenesis and 48 h after transfection on protein level. (B) On day 12, Oilred-O absorbance was assessed to determine adipogenic capacity after MMP11 and PPARg knockdown. (C) The magnitude of PPARg, (D) FABP4, and (E) ATGL mRNA after transfection with siRNAs against control (white square), MMP11 (closed circle) or PPARg (open circle) were assessed by qRT-PCR analyses. For standardization, target gene expression was normalized to the mean of the 2 housekeeping genes: β-actin (ACTB), and TATAbox-binding protein (TBP). *, p < 0.05, **, p < 0.01; ***, p < 0.001.
Figure 4. MMP11 acts as a negative regulator of adipogenesis. (A) MMP11 overexpression efficiency was measured on protein level and compared to cells transfected with empty vector or untreated cells. (B) MMP11 mRNA levels at day 0 and 12 of adipogenesis after MMP11 overexpression and compared to controls. (C) Effects of MMP11 overexpression on adipogenesis were measured by Oilred-O staining at day 12 of adipogenesis. (D) PPARg, (E) FABP4, and (F) ATGL mRNA levels after overexpression of MMP11 (closed circle) compared to an empty vector control (white square) or untreated cells (open circle). For standardization, target gene expression was normalized to the mean of the 2 housekeeping genes: β-actin (ACTB), and TATAbox-binding protein (TBP). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5. MiR125b-5p act as a negative regulator of adipogenesis. (A) MiR125b-5p overexpression (pre-miR125b-5p) efficiency was assessed at day 0 and 12 of adipogenesis (black bars) and compared to pre-ntRNA control (white bar). (B) On day 12, Oilred-O absorbance was measured in order to investigate the effect of miR125b-5p on adipogenesis. (C) MMP11 mRNA levels after miR125b-5p (closed circle) overexpression compared to a pre-ntRNA (white square) control. (D) PPARg, (E) FABP4, and (F) ATGL mRNA levels after miR125b-5p (closed circle) overexpression compared to a pre-ntRNA (white square) control. For standardization, target gene expression was normalized to the mean of the 2 housekeeping genes: β-actin (ACTB), and TATAbox-binding protein (TBP). *, p < 0.05 **, p < 0.01, ***, p < 0.001.
Figure 6. MiR125b-5p downregulation does not affect adipogenesis. (A) MiR125b-5p downregulation efficiency was assessed at day 0 and 12 of adipogenesis (black bars) and compared to pre-ntRNA control (white bar). (B) Oilred-O absorbance was measured at day 12 after adipogenic induction in order to investigate the effect of miR125b-5p on adipogenesis. (C) MMP11 mRNA levels after miR125b-5p (closed circle) downregulation compared to anti-ntRNA (white square) control. (D) PPARg, (E) FABP4, and (F) ATGL mRNA levels after miR125b-5p (closed circle) downregulation compared to an anti-ntRNA (white square) control. For standardization, target gene expression was normalized to the mean of the 2 housekeeping genes: β-actin (ACTB), and TATAbox-binding protein (TBP). *, p < 0.05 **, p < 0.01, ***, p < 0.001.
Figure 7. Schematic overview of the findings. MiR125b-5p directly down-regulates MMP11, which in turn acts as negative regulator of human adipogenesis. Furthermore, miR125b-5p has an anti-adipogenic effect, most likely through the regulation of further unknown pro-adipogenic target(s), which may superimpose the effect of MMP11.
Table 1. Characteristics of the Childhood Adipose Tissue Cohort (n = 100) after stratification in tertiles according to miR125b-5p mRNA levels.