MicroRNA-27 inhibits adipogenic differentiation in orbital fibroblasts from patients with Graves' orbitopathy

Abstract

Background: To investigate the role of microRNA (miR)-27a and miR-27b in adipogenesis in an in vitro model of Graves' orbitopathy (GO).

Methods: Orbital fat tissues were harvested from GO and non-GO participants for primary orbital fibroblast cultures. The expression levels of miR-27a and miR-27b between GO and non-GO orbital fat tissues were compared. During adipogenesis of GO orbital fibroblasts, the expression levels of miR-27a and miR-27b were determined, and the effects of mimics of miR-27a and miR-27b transfection on adipogenesis of GO orbital fibroblast were investigated.

Results: Real time-polymerase chain reaction showed significantly more decreases in miR-27a and miR-27b levels in orbital fat tissues in GO participants than in non-GO participants (p < 0.05). The expression of both miR-27a and miR-27b was highest in orbital fibroblasts at day 0 and declined gradually after the induction of adipogenic differentiation. The expression levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and C/EBPβ were decreased and Oil Red O-stained lipid droplets were lower in GO orbital fibroblasts transfected with miR-27a and miR-27b mimics than in negative controls.

Conclusions: Our results indicated that miR-27a and miR-27b inhibited adipogenesis in orbital fibroblasts from GO patients. Further studies are required to examine the potential of miR-27a and miR-27b as targets for therapeutic strategies.

Fig 1. Comparison of microRNA-27 (miR-27)a and miR-27b expression in Graves’ orbitopathy (GO) and non-GO control participants.

(A) The expression levels of miR-27a and (B) miR-27b were significantly lower in orbital adipose tissues from GO than from non-GO participants (*P<0.05).

Fig 2. MicroRNA-27 (miR-27)a and miR-27b expression during adipogenesis in Graves’ orbitopathy orbital fibroblasts.

(A) The levels of miR-27a and (B) miR-27b were gradually decreased upon adipogenic differentiation. (*P<0.05, **P<0.01).

Fig 3. Cellular microRNA-27 (miR-27)a and miR-27b levels.

(A) The levels of miR-27a and (B) miR-27b increased following transfection of miR-27a and miR-27b mimics in GO orbital fibroblasts. (C) The levels of miR-27a and (D) miR-27b increased following transfection of miR-27a and miR-27b mimics in non-GO orbital fibroblasts. NC, negative control.

Fig 4. Effects of microRNA-27 (miR-27)a and miR-27b mimics on peroxisome-proliferator activated receptor-γ (PPARγ), CCAAT/enhancer binding protein (C/EBP)α, and C/EBPβ mRNA expression in Graves’ orbitopathy (GO) and non-GO orbital fibroblasts.

(A, B) mRNA levels of PPARγ and C/EBPα were increased on day 10 of adipogenesis, and significantly decreased in GO orbital fibroblasts transfected with miR-27a and miR-27b mimics. (C) mRNA levels of C/EBPβ were significantly decreased in GO orbital fibroblast transfected with miR-27b mimics. The mRNA levels of C/EBPβ were decreased in GO orbital fibroblasts transfected with miR-27a mimics, albeit not significantly. (D, E) The mRNA levels of PPARγ and C/EBPα, were increased on 10 day of adipogenesis, and significantly decreased in orbital fibroblasts transfected with miR-27a and miR-27b mimics. (F) The mRNA levels of C/EBPβ were significantly decreased in orbital fibroblasts transfected with miR-27a mimics. The mRNA levels of C/EBPβ were decreased in GO orbital fibroblast transfected with miR-27b mimics, although not significantly. NC, negative control. (*P<0.05, **P<0.01).

Fig 5. Effects of microRNA-27 (miR-27)a and miR-27b mimics on peroxisome-proliferator activated receptor-γ (PPARγ), CCAAT/enhancer binding protein (C/EBP)α, and C/EBPβ protein release in Graves’ orbitopathy orbital fibroblasts.

(A) Representative photo of western blot analysis. (B) Protein levels of C/EBPα, (C) C/EBPβ and (D) PPARγ were increased on 10 day of adipogenesis and significantly decreased in orbital fibroblasts that were transfected with miR-27a and miR-27b mimics (*P<0.05). NC, negative control.

Fig 6. Oil red O staining after adipogenesis on day 10.

(A) No transfection, (B) negative control mimics, (C) microRNA-27(miR-27)a-transfected orbital fibroblasts, (D) miR-27b-transfected orbital fibroblasts. Note that the levels of Oil Red O stained lipid droplets were reduced on day 10 in orbital fibroblasts that were transfected with the miR-27a or miR-27b mimic. (E) The optical density of miR-27a and miR-27b mimics transfected cell lysates showed significantly decreased absorbance at 490 nm (*P<0.01). NC, negative control.