Fig 1: 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.
Fig 2: Protein expression profiles of VDR, CYP27B1, CYP24A1, p21, and E-cadherin in normal, benign, and papillary thyroid cancer (400 × )
Fig 3: Silencing VDR decreases TNF-α, IFN-γ, IL-22, IL-17C, IL-1β and IL-4 expression levels, and increases the 25HVD3 expression level in TNF-α-induced HaCaT cells with stably silenced VDR. The concentrations of (A) TNF-α, (B) 25HVD3, (C) IFN-γ, (D) IL-22, (E) IL-17C, (F) IL-1β and (G) IL-4 were detected by ELISA. ***P<0.001 vs. NC group; #P<0.05, ###P<0.001 vs. TNF-α group. VDR, vitamin D receptor; TNF-α, tumor necrosis factor; IFN-γ, interferon-γ; IL, interleukin; 25HVD3, 25-hydroxyvitamin D3; NC, normal control; LD, low dose; MD, medium dose; HD, high dose; TWP, Tripterygium wilfordii polyglycoside; siRNA, small interfering RNA.
Fig 4: 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.
Fig 5: Silencing VDR using a lentiviral RNAi expression vector in HaCaT cells. HaCaT cells were transfected with the VDR RNAi lentiviruses (VDR544, VDR433, VDR775). (A) The mRNA expression level of VDR was detected by reverse transcription-quantitative polymerase chain reaction assay. (B) The protein expression level of VDR was detected by western blot analysis. GAPDH was used as a loading control. (C) The cell transfection efficiency was determined by infectivity (MOI) detection. Magnification, ×200. Scale bars, 100 µm. *P<0.05, ***P<0.001 vs. NC group. VDR, vitamin D receptor; RNAi, RNA interference; MOI, multiplicity of infection; NC, normal control.
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