Identifying miR-150-5p Binding to PVT1

StarBase v2.0 database (41 (link)) was used to analyze the potential miRNAs that interact with PVT1. miR-150-5p was identified as a potential miRNA. Human PVT1-3′-untranslated region (3′-UTR) and GLUT-1-3′-UTR reporter constructs containing the potential binding site of miR-150-5p, and their identical sequences with a mutation in the seed sequence, which were constructed into pmiR-GLO-basic reporter vector (Promega Corp.). Cells (2×10⁵ cells/well) were plated into 48-well plates for 12 h, co-transfected with the indicated plasmids and miR-150-5p mimics for 24 h. The plasmids (200 ng) were mixed with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), Renilla luciferase plasmids and DMEM for 30 min and then added into the prepared cells for 24 h. Finally, the luciferase activities were measured by the Dual-Luciferase Assay kit (Promega Corp.) according to the manufacturer's instructions. Every sample was tested for three times and the whole experiment was repeated for three times.