Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: TGF-β1 promotes the osteoblastic differentiation of MC3T3-E1 cells. (A) RT-qPCR analysis of the osteogenic-related genes RUNX2, OSX and COL1A1 in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (B) Western blotting of RUNX2, COL1A1-and OSX protein in MC3T3-E1 cells cultured with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (C) ALP activity in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. NS, no significance change; *P<0.05, **P<0.01 as indicated. TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; OSX, osterix; COL1A1 collagen type I α1 chain; ALP, alkaline phosphatase.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Quantitative RT-PCR, Western Blot, Cell Culture, Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: miR-877-3p promotes the osteoblastic differentiation of MC3T3-E1 cells. (A) miR-877-3p expression in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (B) Expression of miR-877-3p was validated in MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitor and their corresponding controls by RT-qPCR. (C) RUNX2, OSX and COL1A1 mRNA expression in MC3T3-E1 cells. (D) MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitor and their corresponding controls were cultured in osteogenic medium. At day 14, the mineralization of differentiated MC3T3-E1 cells was detected with ARS and ALP staining. (E) Transfected MC3T3-E1 cells were cultured in osteogenic medium for 14 days and then mixed with tricalcium phosphate/hydroxyapatite and transplanted into the dorsal region of nude mice for 4-weeks. The results were then evaluated by H&E and Masson's trichrome staining. (F) (F) Transfected MC3T3-E1 cells were cultured in osteogenic medium for 14 days and then mixed with tricalcium phosphate/hydroxyapatite and transplanted into the dorsal region of nude mice for 4-weeks. The Expression expression of miR-877-3p in the xenografts from each group was detected by RT-qPCR analysis. *P<0.05, **P<0.01 and ***P<0.001, as indicated. miR, microRNA; TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; OSX, osterix; COL1A1 collagen type I α1 chain; NC, negative control; ARS, Alizarin Red S; ALP, alkaline phosphatase; H&E, hematoxylin and eosin.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Cell Culture, Staining, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: miR-877-3p inhibits the expression of Smad7. (A) Design of luciferase reporters with the WT Smad7 3′UTR or the site-directed MUT Smad7 3′UTR. (B) Effect of miR-877-3p and miR-NC on luciferase activity in MC3T3-E1 cells transfected with the WT or MUT Smad7 3′UTR. Smad7 expression detected by (C) RT-qPCR and (D) western blotting in MC3T3-E1 cells after culture with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. Smad7 (E) mRNA and (F) protein expression in MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitors and their corresponding controls. *P<0.05, **P<0.01 as indicated. miR, microRNA; WT, wild type; MUT, mutant; UTR, untranslated region; NC, negative control; TGF, transforming growth factor.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Mutagenesis, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: Smad7 is identified as a downstream target of miR-877-3p during the osteoblastic differentiation of MC3T3-E1 cells. mRNA expression of (A) Smad7 and (B) osteoblast differentiation-associated genes in MC3T3-E1 cells transfected with inhibitor-NC, miR-877-3p inhibitor or miR-877-3p inhibitor plus TGF-β1. (C) Expression of RUNX2, Smad7, p-Smad2, t-Smad2, p-Smad3 and t-Smad3 protein in MC3T3-E1 cells transfected with inhibitor-NC, miR-877-3p inhibitor or miR-877-3p inhibitor plus TGF-β1. (D) The mRNA expression of Smad7 was validated in MC3T3-E1 cells transfected with pcDNA-NC or pcDNA-Smad7. The mRNA expression of (E) Smad7 and (F) osteoblast differentiation-associated genes in MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7. (G) Protein levels of Smad7, RUNX2, p-Smad2, t-Smad2, p-Smad3 and t-Smad3 in MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7. (H) ALP activity of MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7 after culture with osteogenic medium for 7 days. (I) MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7 were induced with osteogenic medium. At day 14, the mineralization of differentiated MC3T3-E1 cells was detected with ARS and ALP staining. *P<0.05, **P<0.01 as indicated. miR, microRNA; NC, negative control; TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; p, phospho; t, total; ALP, alkaline phosphatase; ARS, Alizarin Red S.

Article Snippet: For TGF-β1 treatment, recombinant human TGF-β1 (cat. no. CSB-AP003861HU; CUSABIO Technology LLC, Wuhan, China) with final concentration of 4 ng/ml was added to cell culture for 7 or 14 days.

Techniques: Expressing, Transfection, Activity Assay, Staining, Negative Control