MicroRNA-92a-3p regulates the expression of cartilage-specific genes by directly targeting histone deacetylase 2 in chondrogenesis and degradation  G. Mao, Z. Zhang, Z. Huang, W. Chen, G. Huang, F. Meng, Z. Zhang, Y. Kang  Osteoarthritis and Cartilage  Volume 25, Issue 4, Pages 521-532 (April 2017) DOI: 10.1016/j.joca.2016.11.006 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Expression of miR-92a-3p and RNA levels of HDAC2 during chondrogenesis. hMSCs were induced to chondrogenesis with TGF-β3 for 3, 7, 14, 21, and 28 days as indicated (solid lines). Gene expression of miR-92a-3p (a.A), HDAC2 (a.B), Col2a1 (a.C), SOX9 (a.D), Col10a1 (a.E), and Runx2 (a.F) were determined by qRT-PCR. hMSCs cultured without TGF-β3 at corresponding time points served as negative controls (broken lines). U6 and GAPDH were used as internal controls for microRNA and mRNA, respectively. hMSCs treated TGF-β3 on days 3, 7, 14, 21, and 28 were fixed with formalin and stained with alcian blue (b), immunohistochemistry of COL2A1 (c), alizarin red (d), safranin O (e) (magnification, ×400). Data are presented as means ± SDs of six samples.*P < 0.05. Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Expression of miR-92a-3p and RNA levels of HDAC2 during chondrogenesis. hMSCs were induced to chondrogenesis with TGF-β3 for 3, 7, 14, 21, and 28 days as indicated (solid lines). Gene expression of miR-92a-3p (a.A), HDAC2 (a.B), Col2a1 (a.C), SOX9 (a.D), Col10a1 (a.E), and Runx2 (a.F) were determined by qRT-PCR. hMSCs cultured without TGF-β3 at corresponding time points served as negative controls (broken lines). U6 and GAPDH were used as internal controls for microRNA and mRNA, respectively. hMSCs treated TGF-β3 on days 3, 7, 14, 21, and 28 were fixed with formalin and stained with alcian blue (b), immunohistochemistry of COL2A1 (c), alizarin red (d), safranin O (e) (magnification, ×400). Data are presented as means ± SDs of six samples.*P < 0.05. Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 miR-92a-3p regulates the expression of HDAC2, Col2A1, and SOX9 during chondrogenesis. hMSCs were transfected with anti-miR-92a-3p, anti-nonspecific control microRNA (anti-miR-control), and miR-92a-3p or miR-control, and then treated with TGF-β3 to induce chondrogenesis. The expression level of miR-92a-3p (A, E) were estimated by qRT-PCR, while the expression levels of HDAC2 (B, F, I) and Col2a1 (C, G, I) and SOX9 (D, H, I) were estimated by both qRT-PCR and western blotting. U6 and GAPDH were used as endogenous controls. Data were presented as means ± SD of six samples.*P < 0.05. Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 The expression of mature miR-92a-3p and HDAC2 in normal and OA cartilage. Relative miR-92a-3p and HDAC2 mRNA levels in normal and OA cartilages were determined by SYBR green-based qRT-PCR (A and B). U6, and GAPDH were used as endogenous controls. Each dot represents a value from a single experiment of one donor. The bar shows the mean and 95% confidence intervals of the values from six different donors per group. The miR-92a-3p expression levels were determined in normal cartilage and OA cartilage by in situ hybridization (D). HDAC2 protein levels in normal cartilage and OA cartilage were determined by Western blotting and immunohistochemistry using anti-HDAC2 polyclonal antibodies and GAPDH as endogenous controls (C, E). Data shown are representative of results from six normal and OA cartilages (magnification, ×400). Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 miR-92a-3p regulates histone H3 acetylation and SOX9 expression in PHCs. PHCs were transfected with 50 nM miR-control or miR-92a-3p (A) and 100 nM anti-miR-control or anti-miR-92a-3p (B). After 48 h, miR-92a-3p expression was measured by qRT-PCR and normalized to the expression of U6 (A, B), and apoptosis of the cells was detected by annexin V-FITC/propidium iodide double staining with FACS analysis (C, D, E, F). The gene expression levels of ACAN, Col2a1, COMP, dermatopontin, Col9a1 Col10a1, MMP13, ADAMTS-4, ADAMTS-5 and HDAC2 were estimated by qRT-PCR (G, H). Quantitative data are presented as means ± SDs from three independent experiments. GAPDH was used as internal controls for mRNA. *P < 0.05. The acetylation of histone H3 and expression of HDAC2, SOX9, and Runx2 were visualized by western blotting (I). GAPDH and total histone H3 were internal controls for HDAC2, SOX9, Runx2 and ac-H3, respectively. Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 PHCs were transfected with siNC (as control) or siHDAC2. The gene expression levels of ACAN, Col2a1, COMP, dermatopontin, Col9a1 Col10a1, MMP13, ADAMTS-4, ADAMTS-5 and HDAC2 were estimated by qRT-PCR (B). Quantitative data are presented as means ± SDs from six independent experiments. GAPDH was used as internal controls for mRNA, *P < 0.05. The acetylation of histone H3 and expression of HDAC2, SOX9, and Runx2 were visualized by western blotting (A). GAPDH and total histone H3 were internal controls for HDAC2, SOX9, Runx2, and ac-H3, respectively. Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 MiR-92a-3p directly targets HDAC2. HDAC2 was predicted potential targets of miR-92a-3p. Alignment of HDAC2 3′-UTR miR-92a-3p were shown (A). A luciferase reporter carrying the 3′-UTR of HDAC2 or mutant HDAC2 in which the binding site of miR-92a-3p was mutated (Luc-HDAC2-UTR-mut) was introduced into 293T cells along with negative miR-control (NC), miR-92a-3p. The cells were harvested 48 h later for luciferase assays (B). *P < 0.05. Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 a. miR-92a-3p enhances H3 acetylation on COMP, ACAN, and Col2a1 promoter. PHCs were transfected with miR-92a-3p or miR-control (NC) and anti-miR-92a-3p or anti-miR-control and for 24 h before ChIP assay was performed with anti-ac-H3 antibody. ChIP-qPCR showed H3 acetylation of COMP, ACAN, and Col2a1 promoter with and without miR-92a-3p or with and without anti-miR-92a-3p. Data are normalized to input DNA. Values are presented as means ± SD of the results obtained from six samples. *P < 0.05. b. Upstream promoter regions of the COMP, ACAN and COL2A1 genes. The amplified promoter region for ChIP-qPCR with acH3 antibody and primers labeled P is indicated by short arrows. Abbreviations: P, the amplified promoter region for ChIP-qPCR with acH3 antibody and primers; TSS, transcription start site. Osteoarthritis and Cartilage 2017 25, 521-532DOI: (10.1016/j.joca.2016.11.006) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions