2.9. Target Prediction and Dual-Luciferase Reporter Assay

The upstream miRNAs of ANXA6 were predicted with miRDB 6.0 software. A complementary pattern diagram of the miR-24-3p and ANXA6 3′UTR region was created by RNAhybrid 2.1 software. Dual-luciferase reporter assay was performed in chicken embryo fibroblast line (DF-1 cells). Briefly, the DF-1 cells cultured in 48-well plates were co-transfected with reporter vector (ANXA6 3′UTR wild type or mutant type) and mimics NC or miR-24-3p mimics. Following transfection for 48 h, luciferase activity was detected using the Dual Luciferase Reporter Gene Assay Kit (Beyotime, Haimen, China) according to the manufacturer’s instructions.