Psi check 2 luciferase reporter vectors

RNA22 (https://cm.jefferson.edu/rna22/Interactive) and starBase (http://starbase.sysu.edu.cn) databases were used to predict the potential target miRNAs of circFLNA. Among the candidate miRNAs, the top three miRNAs, namely miR-199-3p, miR-296-5p and miR-515-5p, were selected according to their prediction score. Wild-type (WT) and mutant (MUT) putative miR-199-3p binding sites of the 3′-untranslated region (UTR) in circFLNA (Shanghai GenePharma Co., Ltd.), were cloned into psiCHECK2 luciferase reporter vectors (Shanghai GenePharma Co., Ltd.). LN229 cells were seeded at the density of 3×10⁵ cells/well into 6-well plates and co-transfected with 20 µl psiCHECK2-circFLNA-WT or -MUT (10⁸ TU/ml) and 20 µM miR-199-3p mimic or NC mimic using Lipofectamine 2000. The medium was removed with fresh medium at 4 h post-transfection. The relative luciferase activity was detected at 48 h post-transfection using a Dual Luciferase Reporter assay system (cat. no. E1910; Promega Corporation) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.