Luciferase Reporter Assay

Luciferase reporter assay was performed for the detection of direct binding between circATRNL1 and miR-23a-3p and between miR-23a-3p and PTEN. pmir-GLO dual-luciferase vectors (GenePharma, Shanghai, China) containing Renilla luciferase gene and firefly luciferase gene were applied in this experiment. Sequences of circATRNL1, miR-23a-3p, and PTEN were separately cloned into psiCHECK-2 vectors, and mutations were achieved in the binding sites. After 48 h of co-transfection, the luciferase activity was assessed by the dual-luciferase reporter assay (Promega, Madison, WI, USA). Relative firefly luciferase activity was normalized to the corresponding Renilla luciferase internal control.