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. 2022 Nov 11;74(12):1928–1942. doi: 10.1002/art.42285

Figure 6.

Figure 6

Regulation of select extracellular matrix (ECM) genes through a microRNA‐27b‐3p (miR‐27b‐3p)/PPARG/ADAMTS8 signaling axis. PPARG transcript levels (A, D) and protein levels (B, E) were determined in fibroblast‐like synoviocytes (FLS) isolated from osteoarthritis (OA) synovium transfected with either miR‐27b‐3p mimic (Mi) or control mimic (Ctrl M) or transfected with miR‐27b‐3p inhibitor (Inh) or control inhibitor (Ctrl I) and cultured for 48 hours. Results were determined by reverse transcriptase–quantitative polymerase chain reaction (RT‐qPCR) normalized to GAPDH (n = 8) or by Western blot densitometry measured from upper bands appearing at ~58 kd (n = 7). PPARG expression was determined in the synovium of naive mice injected intraarticularly with Mi or Ctrl M (C) or in mice that underwent destabilization of the medial meniscus (DMM) surgery and injected intraarticularly with Inh or Ctrl I (F), as determined by immunohistochemistry. See negative controls in Supplementary Figure 11 (available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10.1002/art.42285). G, Schematic showing transfection of OA FLS with control mimic (Ctrl M) or miR‐27b‐3p mimic (Mi), followed by treatment with rosiglitazone (Rosi) or DMSO as vehicle control or with small interfering RNA (siRNA) targeting ADAMTS8 (AD8) or a negative control siRNA (NC). H and I, OA FLS were transfected with control mimic or miR‐27b‐3p and treated with Rosi/DMSO (H) or ADAMTS8 siRNA/NC siRNA (I). RNA were extracted and assessed by RT‐qPCR for expression of ADAMTS8, COL5A1, and COL1A1. Colored data points in A, D, H, and I highlight biologic replicates (n = 6–9). Scatter plots show mean ± SD, with relative data log‐transformed before analysis by Student's paired 2‐tailed t‐test (A–F) or repeated measures 2‐way analysis of variance followed by Tukey's multiple comparisons post hoc test (H, I). * = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42285/abstract.