miR-150-5p inhibits osteogenic differentiation of fibroblasts in ankylosing spondylitis by targeting VDR

Abstract

Dysregulated microRNAs (miRNAs or miRs) serve potential roles in inflammatory systemic disease, including ankylosing spondylitis (AS). The aim of the present study was to investigate the potential function of miR-150-5p in osteogenic differentiation of AS fibroblasts and its underlying mechanism. The expression of miR-150-5p and vitamin D receptor (VDR) in AS joint capsules and fibroblasts was detected by reverse transcription-quantitative (RT-q)PCR and western blotting. Following overexpression of miR-150-5p, the alteration in osteogenic gene expression was detected by RT-qPCR, western blotting and alkaline phosphatase activity assay, as well as alizarin red staining. The association between miR-150-5p and VDR was confirmed by luciferase assay and rescue experiments were performed. Patients with AS exhibited decreased expression of miR-150-5p in joint capsules. Treatment with bone morphogenic protein 2 (BMP-2) and transforming growth factor-β1 (TGF-β1) led to downregulation of miR-150-5p in AS fibroblasts. Enforced expression of miR-150-5p attenuated osteogenic differentiation of AS fibroblasts. These results demonstrated that miR-150-5p inhibited osteogenic differentiation of AS fibroblasts by targeting VDR. miR-150-5p overexpression decreased osteogenic transformation of fibroblasts by decreasing VDR expression in AS.

Keywords: ankylosing spondylitis; fibroblast; miR-150-5p; osteogenic differentiation; vitamin D receptor.

Figure 1

Downregulation of miR-150-5p in AS joint capsules and fibroblasts. (A) miR-150-5p expression in joint capsule tissues obtained from AS patients and non-AS controls (n=20/group) was determined by reverse transcription-quantitative PCR. (B) miR-150-5p expression in BMP-2 and TGF-β1-treated AS fibroblasts and untreated cells. Data are presented as the mean ± SD. ^**P<0.01 vs. control. miR, microRNA; AS, ankylosing spondylitis; BMP, bone morphogenetic protein; TGF, transforming growth factor.

Figure 2

Overexpression of miR-150-5p suppresses osteogenic differentiation of AS fibroblasts. (A) Transfection efficiency. (B) mRNA expression of Col I, OPN and Runx2 determined by reverse transcription-quantitative PCR. (C) Protein levels of Col I, OPN and Runx2 determined by western blotting. (D) ALP activity. (E) Alizarin red staining for mineralization in BMP-2 and TGF-β1-induced AS fibroblasts transfected with miR-150-5p mimics or miR-NC or untreated cells (magnification, x100). Data are presented as the mean ± SD. ^**P<0.01, ^***P<0.001 vs. control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. miR-NC. miR, microRNA; AS, ankylosing spondylitis; BMP, bone morphogenetic protein; TGF, transforming growth factor; Col I, collagen type I; OPN, osteopontin; ALP, alkaline phosphatase; NC, negative control.

Figure 3

miR-150-5p decreases VDR expression by targeting VDR 3'-UTR. (A) Schematic diagram of the predicted miR-150-5p binding sites to VDR. (B) Luciferase assay in AS fibroblasts transfected with pmirGLO-VDR-WT or -MUT reporters and miR-150-5p mimics or miR-NC. (C) Protein levels of VDR in AS fibroblasts transfected with miR-150-5p mimics or miR-NC. (D) mRNA expression of VDR in joint capsule tissues obtained from patients with AS and non-AS controls (n=20/group) determined by reverse transcription-quantitative PCR. (E) Spearman's correlation analysis of miR-150-5p and VDR mRNA expression in AS joint capsules. (F) VDR protein levels in BMP-2 and TGF-β1-treated AS fibroblasts or untreated cells. Data are presented as the mean ± SD. ^**P<0.01, ^***P<0.001 vs. miR-NC or control. miR, microRNA; AS, ankylosing spondylitis; BMP, bone morphogenetic protein; TGF, transforming growth factor; VDR, vitamin D receptor; UTR, untranslated region; WT, wild-type; MUT, mutant; NC, negative control.

Figure 4

miR-150-5p regulates osteogenic differentiation in AS fibroblasts by downregulating VDR. (A) Transfection efficiency. (B) mRNA expression of Col I, OPN and Runx2. (C) Protein levels of Col I, OPN and Runx2. (D) ALP activity. (E) Alizarin red staining for mineralization in BMP-2 and TGF-β1-treated AS fibroblasts transfected with miR-NC or miR-150-5p mimics and with VDR (magnification, x100). Data are presented as the mean ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Vector or miR-NC; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. miR-150-5p. miR, microRNA; AS, ankylosing spondylitis; BMP, bone morphogenetic protein; TGF, transforming growth factor; VDR, vitamin D receptor; UTR, untranslated region; Col I, collagen type I; OPN, osteopontin; ALP, alkaline phosphatase; NC, negative control.

Figure 5

Schematic diagram of the potential role of miR-150-5p/VDR pathway in BMP-2 + TGF-β1-induced AS osteogenic differentiation. Arrows indicate downregulation. miR, microRNA; AS, ankylosing spondylitis; BMP, bone morphogenetic protein; TGF, transforming growth factor; VDR, vitamin D receptor.