Dual-luciferase reporter assay system

Twenty-four hours before transfection, 5×10⁴ 293T cells/well were cultured in 24-well plates. Cells were then transfected with 40 nM miR-802 mimic or a negative control and co-transfected with 0.8 μg of wild-type 3' UTR-luc or mutant 3'UTR-luc per well using Lipofectamine 2000 according to the manufacturer's instructions. The pGL-4.74 vector (0.1 μg/well) was co-transfected as the endogenous control for luciferase activity. Luciferase activities were assayed 24 h after transfection using a Dual Luciferase Reporter Gene Assay Kit (Beyotime).

The miR-802 promoter was amplified from mouse genomic DNA template and cloned into the pGL-3-basic vector. A promoter containing a mutation in the NF-κB binding site was also inserted into the empty vector. The luciferase reporter assay was performed as described above ⁵⁵.