Pgl luciferase vector

The wild type 3’-UTR of SOX4 (wt SOX4 3’-UTR) contained the complementary
sequences with miR-92a were cloned into pGL luciferase vector (Promega, Madison,
WI, USA). The 3’-UTR of SOX4 containing the mutation of miR-92a recognition
sites (mutated SOX4 3’-UTR) was constructed using a Site-Directed mutagenesis
kit (Agilent Technologies, Santa Clara, CA, USA) and cloned into the pGL
luciferase vector (Promega). Luciferase assay was performed using a dual-Glo
luciferase assay system (Promega).

The binding site between circRNA_103809 and miR-516a-5p was predicted using Circinteractome (https://circinteractome.nia.nih.gov/). The binding site between miR-516a-5p and FBXL18 was predicted using TargetScan7 (http://www.targetscan.org/vert_71/) and miRDB (http://mirdb.org/miRDB/index.html). To perform the luciferase reporter assay, the wild-type or mutant sequence of circRNA_103809 and FBXL18 3ʹUTR was inserted into pGL luciferase vector (Promega, USA). Then, luciferase vector and miR-516a-5p mimics were transfected into EJ cells. After 48 h, the luciferase activity was measured using a dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s protocol. Renilla luciferase activity was normalized control.