Dual-Luciferase Reporter Assay

Binding between circRNA and miRNA, miRNA, and mRNA was validated by luciferase reporter assay. Genechem (Shanghai, China) constructed the luciferase reporter plasmids for the study, in which the 3ʹUTR sequence of circRSU1 or MAP3K8 and its mutants were inserted at the XbaI restriction site, between the Firefly_Luciferase-Renilla_Luciferase vector (hFLuc-XbaL-hRLuc). For luciferase reporter analysis, HEK-293T cells were seeded into 48-well plates and cultured to 50%-70% confluence. A miRNA mimic or mimic-NC (RiboBio, Guangzhou, China) was co-transfected with the specific luciferase reporter plasmid into HEK-293T using Lipofectamine 3000 transfection reagent (ThermoFisher, USA) according to the manufacturer's instructions. Forty-eight hours after incubation, the luciferase activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI). The relative luciferase activity was determined using the value of hRLuc standardized to hFLuc.