Dual-Luciferase Assay Validates circRNA-miRNA Interactions

Interaction analysis was performed using dual-luciferase reporter assay [21 (link)]. The wild-type (WT) sequences of circ_0005526 and TCF4 3ʹUTR were inserted into pmirGLO luciferase plasmid (Promega, Madison, WI, USA), respectively. The constructed plasmids containing miR-142-5p binding sites were named as circ_0005526-WT and TCF4 3ʹUTR-WT. Then miR-142-5p binding sites in circ_0005526 and TCF4 sequences were mutated, and mutant-type (MUT) plasmids (circ_0005526-MUT and TCF4 3ʹUTR-MUT) were used as negative controls. After co-transfection with each plasmid and miR-142-5p or miR-NC for 48 h, luciferase intensity was analyzed through Dual-Luciferase Reporter Detection Kit (E1910; Promega).