Luciferase reporter assay

The luciferase reporter plasmid (Sequences of CircCDK14 or Smad2 and the mutant version were inserted into Xbal restriction sites of Firefly_Luciferase-Renilla_Luciferase vector) were constructed by Genechem (Shanghai, China). In brief, the vector Firefly_Luciferase-Renilla_Luciferase was select for use in this study, and restriction endonuclease XbaI (NEB) was used for vector cleavage to obtain a purified linearized vector. Target gene fragments were amplified and ligated with the linearized vector, followed by transformation of DH5α competent cell. After cultivating with LB medium in culture plates for 12-16 h, the amplified sequence was detected by Sanger Sequencing to verify the consistency with the target genes. The plasmid was extracted and stored at -80 °C. HEK-293T cells were seeded into 24-well plates and cultured to approximately 70% confluence, luciferase reporter plasmid and miR-125a-5p mimic/negative control mimic (RiboBio, Guangzhou, China) were co-transfected into HEK-293T using Lipofectamine 3000 transfection reagent (ThermoFisher) according to the manufacturer's instructions. Forty-eight hours after co-transfection, a dual luciferase reporter assay system (Promega, Madison, WI) was performed to measure the luciferase activity. And firefly luciferase activity was normalized to Renilla luciferase activity for calculation. The map of Firefly_Luciferase-Renilla_Luciferase vector is shown in Table S4.