miRNA Binding Site Validation via Luciferase Assay

StarBase2.0 was used to predict the potential binding sites of miRNAs on DSCAM-AS1 [32 (link)]. Generation of Wild-type (WT) DSCAM-AS1 with potential miR-384 binding sites were inserted into a luciferase reporter vector psi-CHECK-2 (Promega, USA). In addition, a site-directed mutagenesis kit (Tiangen, Beijing, China) was used to generate a mutated form of this vector termed MT-DSCAM-AS1.Co-transfection of CRC cells with luciferase plasmids and miR-384 mimics or miR-NC were carried out followed by culturing for 48 h. Dual-Luciferase Reporter Assay System (Promega) was used to determine the luciferase activities, following the manufacturer’s instructions.