Luciferase reporter assay

The wild-type and mutant 3′-UTR DNA fragments of IFN-γ, MAPK11, and TGF-β2 covering the predicted binding sites of gga-miR-148a-3p were successfully cloned. A total of 1.5 × 10⁶ DF1 chicken fibroblasts/well were seeded in a 6-well plate (Corning, NY, USA). Four hours later, cells at 80% confluence were transfected with plasmid DNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s recommendations. In brief, the target gene with luciferase vector or mutant target gene with luciferase vector was cotransfected with miR-148a-3p into the cells. The pMIR-REPORT β-gal control plasmid (Ambion, Austin, Texas, USA) was also transfected for normalization. After 24 h, the cells were rinsed with 1× PBS, harvested using a scraper, and lysed in 280 µL of 1× luciferase cell culture lysis reagent (CCLR; Promega, Madison, WI, USA). After vortexing for 10–15 s and centrifuging at 14 000 × g for 2 min, the supernatant was used to measure luciferase and β-galactosidase activities in 96-well plates (Corning) in triplicate using Luciferase Assay Systems (Promega) and a β-Galactosidase Enzyme Assay System (Promega), respectively. For the luciferase activity assay, 20 µL/well of the supernatant was mixed with 100 µL/well of Luciferase Assay Reagent in white plates in a dark room, and luciferase activity was immediately measured using a GloMax®-Multi Detection System (Promega). For the β-galactosidase activity assay, 50 µL/well of the supernatant was mixed with 50 µL/well of Assay 2× Buffer in transparent plates followed by incubation at 37 °C for 30 min before the measurement. Reported luciferase activity was normalized to β-galactosidase activity. Each treatment and assay were independently repeated three times.