MicroRNA221-3p modulates Ets-1 expression in synovial fibroblasts from patients with osteoarthritis of temporomandibular joint J. Xu, Y. Liu, M. Deng,

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MicroRNA221-3p modulates Ets-1 expression in synovial fibroblasts from patients with osteoarthritis of temporomandibular joint J. Xu, Y. Liu, M. Deng, J. Li, H. Cai, Q. Meng, W. Fang, X. Long, J. Ke Osteoarthritis and Cartilage Volume 24, Issue 11, Pages 2003-2011 (November 2016) DOI: 10.1016/j.joca.2016.06.011 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 MiRNA expression profile was compared and analyzed using microarray and quantitative real-time PCR in between OA and normal synovial fibroblasts. A: Results of microarray indicated eight up-regulated and six down-regulated miRNAs in OA synovial fibroblasts significantly compared to controls [three controls (one male, two females; mean age: 24.7) and four OAs (one male, three females; mean age: 21.8)]. MiRNA221-3p was the most down-regulated, 5-fold lower in OA synovial fibroblasts. B: Expression of miRNA221-3p was confirmed in control and OA synovial fibroblasts [four controls (two males, two females; mean age: 23.5) and four OAs (one male, three females; mean age: 21.8], P = 0.0286; Box plots, 25th and 75th percentiles; horizontal solid lines, medians; horizontal bars, minimum and maximum. Data were analyzed using Mann–Whitney U test. *: P < 0.05. Osteoarthritis and Cartilage 2016 24, 2003-2011DOI: (10.1016/j.joca.2016.06.011) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Western blot, immunohistochemical staining and FISH were used to detect the expression and distribution of Ets-1 in synovial tissue. A: Western blot. B: Data statistics indicated its expression was about 7-fold higher than that in normal synovial fibroblasts [five OAs (one male, four females; mean age: 26.4), four controls (two males, two females; mean age: 23.5)] (P = 0.0286). C,E: Ets-1 expression was distributed in both synovial lining layer and sublining layer of synovial membrane. D,F: Invaded blood vessels were positive expression for Ets-1. Box plots, 25th and 75th percentiles; horizontal solid lines, medians; horizontal bars, minimum and maximum. Data were analyzed using Mann–Whitney U test. *: P < 0.05. Osteoarthritis and Cartilage 2016 24, 2003-2011DOI: (10.1016/j.joca.2016.06.011) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 To confirm the combination of miR221-3p to Ets-1 mRNA, miRNA221-3p mimics, inhibitor or negative control was transfected into OA synovial fibroblast. Ets-1 3′-UTR/mutant reporter system was also co-transfected with miRNA221-3p mimics or negative control in 293E cells for 48 h. A: mRNA level of Ets-1 expression 24 h after transfection of miRNA221-3p mimics and inhibitor. B: Protein expression of Ets-1 48 h after transfection. Note: Expression levels were uniformly set to a value of one in negative control. The data were shown as mean ± SD of n = 3 patients (three females; mean age: 21). C: Structure of 3′-UTR luciferase reporter system. Predicted result of miRNA221-3p targeting Ets-1 mRNA 3′-UTR and mutant sequence. D: Relative luciferase activity was significantly reduced in 3′-UTR+miRNA221-3p group compared with other groups (P = 0.0002). Values were shown as mean ± SD of n = 5 and analyzed using one-way ANOVA test. *P < 0.05. NC: negative control. Osteoarthritis and Cartilage 2016 24, 2003-2011DOI: (10.1016/j.joca.2016.06.011) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 To explore effect of miRNA221-3p on MMPs expression, transfection was divided into following four groups: negative control, miRNA221-3p mimics, miRNA221-3p inhibitor, miRNA221-3p inhibitor+specific Ets-1 siRNA. Protein levels were detected after 72 h. A: Western blot was used to detect protein expression of MMP1, MMP3 and MMP9. B: MMP1 expression. C: MMP3 expression. D: MMP9 expression. Box plots, 25th and 75th percentiles; horizontal solid lines, medians; horizontal bars, minimum and maximum. Data were analyzed using Mann–Whitney U test (n = 4 patients, one male, three females; mean age: 21.8). *: P < 0.05 compared with NC group. NC: negative control. Osteoarthritis and Cartilage 2016 24, 2003-2011DOI: (10.1016/j.joca.2016.06.011) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 OA synovial fibroblast was stimulated by IL-1β for indicated periods of time with gradient doses. MiRNA and protein was extracted. A: miRNA221-3p expression after IL-1β stimulation at a different dose for 24 h. B: miRNA221-3p expression after IL-1β stimulation in different time. C: Ets-1 protein expression after IL-1β stimulation at a different dose for 24 h. D: Protein expression of Ets-1 after IL-1β stimulation in different time. Note: Values were uniformly set to a value of one initially. Values were shown as mean ± SD of n = 3 patients (three females; mean age: 21). Osteoarthritis and Cartilage 2016 24, 2003-2011DOI: (10.1016/j.joca.2016.06.011) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions