Dataset Information

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 PPAR?, CCAAT/enhancer binding protein (C/EBP)? and C/EBP? 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.

SUBMITTER: Jang SY

PROVIDER: S-EPMC6695164 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

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

Jang Sun Young SY Chae Min Kyung MK Lee Joon H JH Lee Eun Jig EJ Yoon Jin Sook JS

PloS one 20190815 8

<h4>Background</h4>To investigate the role of microRNA (miR)-27a and miR-27b in adipogenesis in an in vitro model of Graves' orbitopathy (GO).<h4>Methods</h4>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 mimi ...[more]

PMID: 31415657

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Project description:PURPOSE:Chemokines are involved in the pathogenesis of various autoimmune diseases, including Graves' orbitopathy (GO), but comprehensive analyses of the dynamics of these cytokines and their receptors in such diseases remain lacking. In this study, we investigated the expressions of chemokines and their receptors during adipogenesis and inflammation in primary cultured orbital fibroblasts from patients with GO. METHODS:The messenger RNA (mRNA) expression levels of chemokines were compared between GO (n = 6) and non-GO (n = 5) orbital tissues by real-time polymerase chain reaction. After adipogenesis was induced in primary cultured orbital fibroblasts from patients with GO (n =5) and following stimulation with interleukin (IL)-1? and tumor necrosis factor (TNF)-?, the mRNA expression levels of chemokines and their receptors were analyzed. RESULTS:Chemokines were significantly downregulated in GO orbital tissues compared to non-GO orbital tissues (p < 0.05). Adipogenesis resulted in a strong increase in mRNA expression levels of chemokines and their receptors at an early stage (day 1); however, expression levels started to decrease thereafter and, eventually, decreased to below basal levels at the end of adipogenesis (day 10). Following stimulation with IL-1? and TNF-?, the mRNA expression levels of chemokines and their receptors increased, showing different responses to various proinflammatory cytokines. CONCLUSIONS:Chemokines were strongly upregulated in the early phase of adipogenesis before decreasing continuously until the end of adipogenesis. Also, overt mature GO tissues showed reduced mRNA expression of chemokines compared to controls, which might indicate the existence of a shorter window for effective medical inflammatory treatment. The heightened levels of chemokines and their receptors observed after stimulation with IL-1? and TNF-? suggest a crucial role of proinflammatory cytokines in the pathogenesis of GO and, further, support the idea that chemokines could be used as biomarkers of GO activity.

Project description:PurposeBruton's tyrosine kinase (BTK) is an essential protein in B-cell antigen receptor (BCR) signaling pathway and is known to be related to pathogenetic effect on B-cell related malignancies and various autoimmune diseases. In this study, we investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK inhibitor in the pathogenesis of Graves' orbitopathy (GO) in in vitro model.MethodsExpression of BTK in orbital tissues from GO and normal control subjects were evaluated by real-time polymerase chain reaction (PCR). Primary cultured orbital fibroblasts from each subject were exposed to ibrutinib and stimulated with interleukin (IL)-1β or insulin like growth factor (IGF)-1. Production of inflammatory cytokines was evaluated by real time PCR and enzyme-linked immunosorbent assays (ELISA). The downstream transcription factors were also determined by western blot assays.ResultsThe expression of BTK in GO tissues were significantly higher than in healthy controls. After stimulation of GO orbital fibroblasts with IL-1β or IGF-1, BTK mRNA and phosphorylated (p)- BTK protein expression was also enhanced. Ibrutinib reduced the expression of BTK mRNA and proteins of p-BTK, and inhibited the IL-1β- and IGF-1-induced production of proinflammatory cytokines including IL-6, IL-8 and COX-2 in both GO and normal cells. Ibrutinib also significantly attenuated phosphorylation of Akt, p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) in IL-1β stimulated GO cells and Akt, JNK, and NF-κB in IL-1ß stimulated normal cells.ConclusionsBTK expression is enhanced in GO tissue and orbital fibroblasts. Ibrutinib, a BTK inhibitor suppresses proinflammatory cytokine production as well as phosphorylation of Akt and NF-κB protein. Our results suggest the potential role of BTK in GO inflammatory pathogenesis and possibility of a novel therapeutic target of GO.

Project description:BackgroundThe proprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated in the pathogenesis of inflammatory diseases. We sought to investigate the role of PCSK9 in the pathogenesis of Graves' orbitopathy (GO) and whether it may be a legitimate target for treatment.MethodsThe PCSK9 was compared between GO (n=11) and normal subjects (n=7) in orbital tissue explants using quantitative real-time PCR, and in cultured interleukin-1β (IL-1β)-treated fibroblasts using western blot. Western blot was used to identify the effects of PCSK9 inhibition on IL-1β-induced pro-inflammatory cytokines production and signaling molecules expression as well as levels of adipogenic markers and oxidative stress-related proteins. Adipogenic differentiation was identified using Oil Red O staining. The plasma PCSK9 concentrations were compared between patients with GO (n=44) and healthy subjects (n=26) by ELISA.ResultsThe PCSK9 transcript level was higher in GO tissues. The depletion of PCSK9 blunted IL-1β-induced expression of intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2) in GO and non-GO fibroblasts. The levels of activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated forms of Akt and p38 were diminished when PCSK9 was suppressed in GO fibroblasts. Decreases in lipid droplets and attenuated levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein β (C/EBPβ), and leptin as well as hypoxia-inducible factor 1α (HIF-1α), manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1) were noted when PCSK9 was suppressed during adipocyte differentiation. The plasma PCSK9 level was significantly higher in GO patients and correlated with level of thyrotropin binding inhibitory immunoglobulin (TBII) and the clinical activity score (CAS).ConclusionsPCSK9 plays a significant role in GO. The PCSK9 inhibition attenuated the pro-inflammatory cytokines production, oxidative stress, and fibroblast differentiation into adipocytes. PCSK9 may serve as a therapeutic target and biomarker for GO.

Dataset Information

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

Background

Methods

Results

Conclusions

Publications

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

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