1. MicroRNA-320 regulates matrix metalloproteinase-13 expression in chondrogenesis and interleukin-1β-induced chondrocyte responses 
F. Meng, Z. Zhang, W. Chen, G. Huang, A. He, C. Hou, Y. Long, Z. Yang, Z. Zhang, W. Liao 
Osteoarthritis and Cartilage 
Volume 24, Issue 5, Pages (May 2016)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Expression of miR-320 during chondrogenesis. ATDC5 cells were induced to undergo chondrogenesis for the indicated amount of time. Gene expression and glycosaminoglycan deposition in treated cells were compared with the levels in untreated cells by qRT-PCR. The expression levels of miR-320 (A), chondrogenic markers (Col2a1 and Sox9) (B, C), and hypertrophic markers (Col10a1, Runx2, and Mmp-13) (D–F) were estimated. Rnu6b and Gapdh were used as endogenous controls. ATDC5 cells treated for day 3, 7, 14, 21, and 28 were fixed with formalin and stained with Alcian Blue to detect glycosaminoglycan deposition (G–H). Scale bar = 200 μm (H). Data represent the mean ± standard error (SE) of three experiments; *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
miR-320 regulates the expression of MMP-13 and COL2A1 during chondrogenesis. ATDC5 cells were transfected with anti-miR-320-3p, anti-nonspecific control microRNA (anti-miR-Control), and miR-320 or miR-Control, and then treated with insulin, transferrin, and selenous acid (ITS) to induce chondrogenesis. The expression level of miR-320 (A, E) and Col10a1 (D, H) were estimated by qRT-PCR, while the expression levels of Mmp-13 (B, F, I) and Col2a1 (C, G, L) were estimated by both qRT-PCR and western blotting. Western blots were quantified by integrated density analysis from three experiments (J, K, M, N). Rnu6B and Gapdh were used as endogenous controls. Data represent the mean ± standard error (SE) of three experiments; *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Expression of miR-320 and MMP-13 in normal and osteoarthritic human cartilage and in IL-1β-stimulated mouse chondrocytes. The relative miR-320 and MMP-13 levels in normal and OA cartilage were determined by qRT-PCR (A, B). RNU6B/GAPDH was used as an endogenous control. Symbols represent individual cartilage donors; bars show the mean and 95% confidence interval of each group. MMP-13 protein levels were determined in normal cartilage and OA cartilage by immunohistochemistry (C). miR-320 expression levels were determined in normal cartilage and OA cartilage by in situ hybridization (D). Representative results from three normal and three OA cartilages are shown (magnification, 200×). PMCs were deprived of serum for 24 h, and then left untreated or treated with various concentrations of IL-1β for the indicated times (E–J). The relative expression levels of miR-320 (E, H) and Mmp-13 (F, I) were assessed by qRT-PCR; Rnu6b and Gapdh were used as endogenous controls. IL-1β-induced MMP-13 production in PMCs was determined by ELISA, after 3, 6, 12 (G), or 24 h (J) of stimulation with IL-1β. Scale bar = 100 μm (C, D). Data represent the mean ± standard error (SE) of three experiments. P values were computed compared to non-stimulated controls; *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Negative correlation between the expression patterns of miR-320 and Mmp-13 regulated by IL-1β-induced signaling pathways. PMCs were pretreated for 2 h with MAPK inhibitors (SB203580, PD98059, or SP600125) or an NF-κB inhibitor (SN50), and then left unstimulated or stimulated with IL-1β for 6 (A, B) or 24 h (C). PMCs were analyzed by qRT-PCR for miR-320 (A) and Mmp-13 mRNA expression (B) and by ELISA for MMP-13 protein production in culture supernatants (C). Rnu6b and Gapdh were used as endogenous controls for qRT-PCR. Data represent the mean ± standard error (SE) of three experiments. P values were computed vs non-stimulated controls* or IL-1β-stimulated controls#; (*,#) P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Effect of miR-320 on IL-1β-induced MMP-13 expression. PMCs were transfected with miR-320 or nonspecific control microRNA (miR-Control), or with anti-miR-320-3p or anti-miR-Control, and then left unstimulated or stimulated with IL-1β for 6 or 24 h for qRT-PCR analysis or protein analysis, respectively. At 48 h after transfection with miR-320 or miR-Control (A, B, C, G, H, I), or with anti-miR-320-3p or anti-miR-Control (D, E, F, J, K, L), the expression levels of miR-320 (A, D) and Mmp-13 (B, E) were determined by qRT-PCR. Rnu6b and Gapdh were used as endogenous controls. MMP-13 protein production was analyzed by ELISA and western blotting (C, G, F, and J). Western blots were quantified by integrated density analysis from three experiments (H, I, K, L). Data represent the mean ± standard error (SE) of three experiments. P values were computed vs non-stimulated controls* or IL-1β-stimulated controls#; (*,#) P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
miR-320-3p targets the 3′-untranslated region (3′-UTR) of MMP-13 mRNA. Sequence of the putative miR-320-3p binding site within the 3′-UTR of MMP-13 mRNA (A). The MMP-13 3′-UTR (MMP-13) reporter plasmid (B) or mutated-type MMP-13 3′-UTR (MMP-13-Mut) reporter plasmid (C) was co-transfected with miR-320-3p or nonspecific control microRNA (NC) into the cells. Cells transfected with only MMP-13 or MMP-13-Mut were used as controls. The fluorescence intensity was measured. Data represent the mean ± standard error (SE) of three experiments; *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

8. Supplementary Fig. 1
Expression of miR-140 during chondrogenesis. Data represent the mean ± standard error (SE) of three experiments; *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

9. Supplementary Fig. 2
Expression of miR-27b in interleukin-1β (IL-1β)-stimulated mouse chondrocytes. Data represent the mean ± standard error (SE) of three experiments; *P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions