Luciferase Reporter Assays
The circ-000543 wild sequence, ZNF268-3′UTR wild sequence and their mutations with or without miR-567 binding sites were synthesized and then subcloned into luciferase reporter vectors psiCHECK-2, which was bought from Promega (Madison, WI, USA). HEK293T cells were implanted in 24-well plates at the density of 3×10
4 cells/well in triplicate. Then, miR-567 or NC mimic were cotransfected with corresponding psiCHECK-2 plasmids. After co-transfection for 48 h, dual-luciferase reporter assay system that provided by Promega was proceeded to conduct the luciferase reporter assays according to the manufacturer’s instructions. Relative luciferase activity was subsequently normalized to the Renilla luciferase internal control.