Fig 1: miR-672-5p Represses Smurf1 to Promote Osteoblast Differentiation(A) Stem-loop structure of miR-672 predicted by miR-base (the mature miR-672-5p sequence is shown in green). (B and C) Expression (qPCR, in triplicate) of miR-672-5p in different tissues (B) and cells, i.e., in osteoblasts during proliferation, differentiation, or mineralization (C). Data are mean ± SE. *p < 0.05 and ***p < 0.001 compared with proliferation stage of osteoblasts. (D–F) Effect of transfection of osteoblasts with the mimic (miR-672-5p), inhibitor (anti-miR-672-5p), and miR-C (control) on ALP (alkaline phosphatase) activity (OD, n = 8) (D) and mineralization (OD, n = 4) (E). (F) Representative wells showing alizarin-positive colony (Cfu-ob) formation in osteoblast cell cultures on day 15 in osteogenic medium. Data are mean ± SE. *p < 0.05 and **p < 0.01 compared with miR-C. (G) Identification of miR-672-5p target gene in osteoblasts and computational analysis was performed for the complementarities of miR-672-5p to the 3′ UTR of Smurf1 and (H) schematic presentation of the reporter plasmid used to illustrate the effect of Smurf1 3′ UTR on luciferase activity. CMV, cytomegalovirus promoter; Luc, luciferase; RLuc, renilla luciferase. (I) Effect of miR-672-5p overexpression on a dual luciferase reporter plasmid containing Smurf1 3′ UTR was analyzed. Cells were co-transfected with the WT-pEZX-MT06-Smurf1 or an empty vector (NC, negative control) and miR-672-5p or miR-C. Firefly and renilla luciferases were measured in the cell lysate. Data are mean ± SE of three independent measurements. **p < 0.01 compared with miR-C. (J) Expression (qPCR, in triplicate) of Smurf1 in osteoblasts during proliferation, differentiation, or mineralization. Data are mean ± SE. *p < 0.05 compared with proliferation stage of osteoblasts. (K) Transfection of osteoblasts with the mimic (miR-672-5p) enhanced the release of BMP2 in conditioned medium (BMP2 ELISA). Data are mean ± SE of three independent experiments. *p < 0.05 compared with miR-C. (L) Effect of miR-672-5p or scrambled miR-C on Runx2 promoter activity using a luciferase (Luc) reporter. Data are mean ± SE of three independent experiments. *p < 0.05 compared with miR-C. (M) qPCR (in triplicate) was performed for Smurf1 (a direct target gene of miR-672-5p) or the osteoblast differentiation genes, including Runx2, BMP2, Smad1, and ALP after 48 h of transfection. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with miR-C. (N) Western blot analysis was done for Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1000), BMP2 (ab14933-Abcam; 1:1000), and Smad1 (ab63356-Abcam; 1:1,000) after 48 h of transfection. β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were horseradish peroxidase (HRP) conjugated (Sigma-Aldrich). (O–S) miR-672-5p represses Smurf1, which promotes the upregulation of Runx2 and osteoblast differentiation. (O) ALP activity (OD, n = 8), (P) Cfu-ob formation, (Q) mineralization (OD, n = 4), and (R) mRNA expression of Runx2, BMP2, Smurf1, ALP, and Smad1 (qPCR, in triplicate), and (S) protein expression of Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1000), and BMP2 (ab14933-Abcam; 1:1,000) in either control osteoblasts or transfected with siRNA against Smurf1 or scrambled siRNA (Si-Scr). β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were HRP conjugated (Sigma-Aldrich). Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with miR-C or control. Other comparisons, ˆp < 0.05, ˆˆp < 0.01, and ˆˆˆp < 0.001 as shown.
Fig 2: miR-672-5p Treatment Represses Smurf1 In Vivo to Promote Osteoblastogenesis(A) ALP activity (OD, n = 6), (B) mineralization (OD, n = 6), and (C) representative wells of Cfu-ob formation. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group, or as shown ˆˆp < 0.01. mRNA expression (qPCR, in triplicate) of Smurf1 (D), Runx2 (E), and BMP2 (F) and protein expression (G) (western blotting) of Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1,000), and BMP2 (ab14933-Abcam; 1:1,000) in osteoblasts from sham-operated (Sham) or ovariectomized (OVx) mice that were given PBS (0.2 mL), scrambled miR (miR-C, 7.0 mg kg−1), or miR-672-5p (7.0 mg kg−1). β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were HRP conjugated (Sigma-Aldrich). Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group. See also Figures S2D–S2F for in vitro actions of microRNA-672-5p on osteoclastogenesis from bone marrow cell cultures.
Fig 3: Hes1 is directly bound to the promoters of Runx2 and Bmp2. A chromatin immunoprecipitation (ChIP) assay was performed using chromatin isolated from D1 cells; anti-Hes1 antibodies were used for immunoprecipitation. After 24-h IS treatment (antibodies used in ChIP: those against IS + Hes1) or no treatment (antibodies used in ChIP: those against Hes1 only), DNA from D1 cells was analyzed through quantitative PCR performed using primers targeting the Hes1-binding sites in the promoters of (A) Runx2 and (B) Bmp2. Immunoglobulin (Ig) G served as the negative control, whereas the input (total DNA extract) served as the positive control. The experiments were performed in triplicate. Data are presented as mean ± standard deviation values (** p < 0.01, and *** p < 0.001; analysis of variance).
Fig 4: miR-181c-5p represses BMP2-induced SMAD7 expression. (a) Fluorescence microscopy observation of BMSCs after 3 days of infection with BMP2 overexpressing adenovirus (scale bar = 25 μm). (b) BMP2 mRNA and protein levels in BMSCs after BMP2 overexpression determined by RT-qPCR and Western blot analysis. (c) SMAD7 mRNA level in BMSCs after BMP2 overexpression determined by RT-qPCR. (d) Fluorescence microscopy observation of BMSCs after 3 days of infection with miR-181c-5p adenovirus (scale bar = 25 μm). (e) miR-181c-5p expression after overexpression of miR-181c-5p in BMSCs determined by RT-qPCR. (f) SMAD7 expression in BMSCs after overexpression of miR-181c-5p determined by RT-qPCR. (g) mRNA level of SMAD7 in BMSCs treated with Ad-BMP2 alone or combined with Ad-miR-181c-5p determined by RT-qPCR. (h) Protein level of SMAD7 in BMSCs treated with Ad-BMP2 alone or combined with Ad-miR-181c-5p measured by Western blot analysis. ∗p < 0.05. All experiments were repeated for three times.
Fig 5: A diagrammatic view of NOTCH1/LSD1/BMP2 signaling network in miR-137-mediated differentiation of hASCs towards osteoblastic lineage. In this complex network model, miR-137 modulates NOTCH1-HES1, LSD1, and BMP2-SMADs pathways simultaneously. Depending on the positive feedback loop between NOTCH1 and BMP2, as well as the negative reciprocal relationship between LSD1 and NOTCH1 or BMP2, the impacts of miR-137 on the above three signaling pathways are strengthened and the NOTCH1/LSD1/BMP2 network regulates the osteogenesis of hASCs coordinately (red lines represent downregulation and green lines represent upregulation)
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