Figure 3
MiR-146a inhibits the expression of cartilage matrix-associated genes. (a) qPCR analysis of miR-146a levels in knee joint cartilage of C57BL/6 male mice receiving OA surgery, or sham operation at the age of 12 weeks. Samples were collected at 4 weeks after surgery. Sham, n=11; OA, n=13. (b) qPCR analysis of pro-inflammatory cytokines and MMPs mRNA expression in articular cartilage pooled from 6 sham-operated C57BL/6 mice or 13 mice receiving OA surgery. (c) Immunostaining of mouse primary articular chondrocytes with an antibody against type II collagen. Scale bar, 50 μm. (d) qPCR analysis of miR-146a levels in mouse primary articular chondrocytes stimulated with IL-1β (10 ng/ml), TNF-α (10 ng/ml), IL-6 (10 ng/ml), IL-17 (50 ng/ml), IFN-γ (10 ng/ml) or TGF-β (10 ng/ml) for 24 h (n=3 for each treatment). *P<0.05, **P<0.01 versus mock. (e and f) Mouse primary chondrocytes were infected with Lenti-mimic NC and Lenti-mimic-146a (e), or Lenti-inhibitor NC and Lenti-inhibitor 146a (f) at 20 MOI for 24 h, then incubated with IL-1β, TNF-α, IL-17 or left untreated for an additional 48 h (n=3 for each treatment). The protein amounts of Col2a1 and Sox9 were determined by western blot. For quantification, protein expression was normalized by the protein amount in the first lane using Image J 1.42 software (NIH, Bethesda, MD, USA). Data are representative of three independent experiments in d-f. (f and g) qPCR analysis of mRNA expression of Sox9 and Col2a1 in knee joint cartilage pooled from four WT mice and eight miR-146a KO mice harvested at 4 weeks after DMM surgery. The relative mRNA expression levels were normalized to the expression of GAPDH. Data are the mean±S.D. *P<0.05, **P<0.01 versus control group (Student’s t-test) in (d) and Mann–Whitney test in a, b, g and h
