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. Author manuscript; available in PMC: 2018 Mar 1.
Published in final edited form as: J Bone Miner Res. 2016 Oct 28;32(3):461–472. doi: 10.1002/jbmr.3005

Fig. 4.

Fig. 4

miR-221-3p inhibits osteoblast differentiation. (A) Expression of miR-221-3p is upregulated in FLS treated with TNF (10 ng/mL, 72 hours). Relative expression of miRNA was examined by qPCR (error bars are SD). Expression of miR-221-3p is normalized to U6. (B) Relative expression of miR-221-3p in whole synovial tissue is validated by qPCR in synovial RNA samples (n =2 pools each with technical replicates, error bars are SD). Expression of miR-221-3p is normalized to U6. (C) Expression of miR-221-3p in FLS-derived exosomes is analyzed by qPCR. Expression of miR-221-3p is normalized to a spike in control (cel-miR-39). *p ≤0.05 (Experiment is performed with three independent biologic replicates, error bars are SD). (D) Alkaline phosphatase (red) and Von Kossa (black) staining of calvarial osteoblasts transfected with control miRNA, miR-221-3p, and anti-miR-221-3p. Experiment is repeated in triplicate. (E) qPCR of early and mature osteoblast markers and a readout of Wnt signaling, Tcf-1. (error bars are SD, performed in triplicate), **p ≤0.01, ***p ≤0.001. (F) Western blot for a target of miR-221-3p, Dkk2. Expression of Dkk2 protein in calvarial cells transfected with control miRNA and miR-221-3p (error bars are SD), *p ≤0.05. (G) Dkk2 3′UTR luciferase reporter activity. MC3T3 cells are co-transfected with Dkk2 3′UTR or mutated Dkk2 3′UTR reporter along with negative control or miR-221-3p. Firefly luciferase activity is normalized with renilla luciferase activity and expressed as relative luciferase activity (control =1). (error bars are SD, performed in five wells each. Experiment is repeated in triplicate), *p ≤0.05. (H) Schematic: synovium-derived miR-221-3p and its potential role in regulating osteoblast differentiation. TNF upregulates expression of miR-221-3p in FLS. Upregulation of miR-221-3p downregulates Dkk2 expression in osteoblasts. Downregulating Dkk2 expression would contribute to the suppression of osteoblast differentiation and inhibition of bone repair at erosion sites. FLS=fibroblast-like synoviocyte.