miR-6402 targets Bmpr2 and negatively regulates mouse adipogenesis

Abstract

Obesity is characterized by macrophage infiltration into adipose tissue. White adipose tissue remodelling under inflammatory conditions involves both hypertrophy and adipogenesis and is regulated by transcription factors, which are influenced by bone morphogenetic protein (BMP) signalling. MicroRNAs (miRNAs) regulate gene expression and are involved in obesity-related processes such as adipogenesis. Therefore, we identified differentially expressed miRNAs in the epididymal white adipose tissue (eWAT) of mice fed a normal diet (ND) and those fed a high-fat diet (HFD). The expression of miR-6402 was significantly suppressed in the inflamed eWAT of HFD-fed mice than in ND-fed mice. Furthermore, Bmpr2, the receptor for BMP4, was identified as a target gene of miR-6402. Consistently, miR-6402 was downregulated in the inflamed eWAT of HFD-fed mice and in 3T3-L1 cells (preadipocytes) and differentiated 3T3-L1 cells (mature adipocytes) , and BMPR2 expression in these cells was upregulated. Adipogenesis was induced in WAT by BMP4 injection (in vivo) and in 3T3-L1 cells by BMP4 stimulation (in vitro), both of which were inhibited by miR-6402 transfection. Inflamed eWAT showed higher expression of BMPR2 and the adipogenesis markers C/EBPβ and PPARγ, which was suppressed by miR-6402 transfection. Our findings suggest that miR-6402 is a novel anti-adipogenic miRNA that combats obesity by inhibiting the BMP4/BMPR2 signalling pathway and subsequently reducing adipose tissue expansion.

Keywords: Adipocyte; adipogenesis; bone morphogenetic protein receptor type 2; miR-6402; microRNA.

Figure 1.

miR-6402 is suppressed in the obese mouse adipose tissues and tnf-α-stimulated adipocytes. (a) Heatmap of downregulated miRNAs in eWAT after HFD feeding in comparison with the findings in nd-fed mice. (b–f) qPCR analyses were performed to assess miR-6402 expression in eWAT (b), differentiated 3T3-L1 cells co-cultured with RAW264.7 cells (c), or RAW264.7 cells (d) stimulated with LPS for the indicated times, and undifferentiated (e) or differentiated (f) 3T3-L1 cells stimulated with tnf-α (5 ng/mL) for 4 h. The bar graphs represent the mean ± S.D. (n = 3 for each group). *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated bars of the two groups (b, e, and f; Student’s t-test; c and d, Tukey – Kramer HSD test).

Figure 2.

Bmpr2 is the target of miR-6402 and is upregulated in inflamed adipocytes. Undifferentiated (a and b) and differentiated (c and d) 3T3-L1 cells were transfected with negative control microRNA (control) or miR-6402 mimics for 24 h. Significant expression of miR-6402 was observed in undifferentiated and differentiated 3T3-L1 cells (supplementary fig. S3a and b), indicating successful transfection of miR-6402. Expression of the indicated potential miR-6402 target genes predicted by TargetScan was measured by qPCR (a). BMPR2 expression in undifferentiated 3T3-L1 cells transfected with control and miR-6402 mimics was analysed by western blotting (b), BMPR2 expression in differentiated 3T3-L1 cells transfected with control and miR-6402 mimics was analysed by qPCR (c) and western blotting (d). BMPR2 expression was measured by qPCR (e and g) and western blotting (f and h) using undifferentiated (e and f) or differentiated (g and h) 3T3-L1 cells stimulated with tnf-α (5 ng/mL). β-actin was used as the loading control. Original whole-membrane and repeated images are shown in supplementary fig. S4, S5, S6 and S7. The bar graphs represent the mean ± S.D. (n = 3–4 for each group). *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated bars of the two groups (a – e and g, Student’s t-test; f and h, Tukey – Kramer HSD test).

Figure 3.

miR-6402 inhibits BMP4-induced adipogenesis. (a) Oil red O staining on day 0, 3, and 6. 3T3-L1 cells were cultured to 50–70% confluence and transfected with miR-6402 mimic. After 24 h, the cells were stimulated with a combination of differentiation media in the presence or absence of BMP4 (10 ng/mL). Scale bar: 100 µm. (b – d) expression of BMPR2, C/EBPβ, and PPARγ by qPCR (b and c) or western blotting (d) in eWAT from ND- or hfd-fed mice. Original whole-membrane images of (d) are shown in supplementary fig. S8. β-actin was used as the loading control. Data represent the mean ± SD (n = 3–4 for each group). *p < 0.05, **p < 0.01 (Student’s t-test).

Figure 4.

miR-6402 inhibits BMP4-induced adipogenesis in vivo. (a–e) BMP4 was injected into eWAT together with control or miR-6402 mimic. Two out of the seven mice were used for immunohistochemical staining of BMPR2 and hematoxylin-eosin staining (a; mouse No. 1 and 2), and the other five mice were used for analysis of BMPR2, C/EBPβ, and PPARγ expression by western blotting (e; mouse No. 3–7). Four different images from mice 1 and 2 (i.e. 8 images from two mouse eWAT samples) were obtained and analyzed for the immunohistochemical staining intensity of BMPR2 (b), frequency and mean cell size of adipocytes (c), and number of adipocytes (d). Representative image sets are shown in (a). Scale bar; 100 µm. Other images are shown in supplementary fig. S10. Western blotting results are shown on the far left in (e), and these analyzed data are shown as three bar graphs on the right. β-actin was used as a loading control. Original whole-membrane images are shown in supplementary fig. S11. The bar graphs represent the mean ± S.D. *p < 0.05, ***p < 0.001 between the indicated bar of two groups (Student’s t-test).