Pmirglo dual luciferase target vector

Utilizing the bioinformatics tool TargetScan, miRWalk, and miRDB, we predicted the potential binding sites between miR-92b-3p and the ELK4 3′UTR sequence. The 3′UTR fragment of ELK4 containing miR-92b-3p binding site wild-type (WT) or mutant (MUT) was cloned into a pmir-GLO Dual-luciferase Target Vector (Promega, Madison, WI, USA). The pmir-GLO vector (WT fragments or MUT fragments) and miR-92b-3p mimic or the negative control were transfected into the 293T cells using Lipofectamine 2000 (Invitrogen, USA). The Dual-Luciferase Reporter Assay System (Promega, USA) was implemented to measure luciferase activity 48 h after transfection according to the guidelines. The co-expressed Renilla luciferase activity was used to normalize the firefly luciferase activity.

LINC00514 fragments harboring the WT and mutant miR-708-binding sites were designed and chemically synthesized by Shanghai GenePharma Co., Ltd. The WT and MUT LINC00514 fragments were cloned into the pmirGLO Dual-luciferase Target Vector (Promega Corporation, Madison, WI, USA) to produce WT-LINC00514 and MUT-LINC00514 reporter plasmids, respectively. After culture in 24-well plates, WT-LINC00514 or MUT-LINC006514 was cotransfected into cells with miR-708 mimic or miR-NC using Lipofectamine® 2000. At 48 h after transfection, luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega Corporation). Renilla luciferase activity was normalized to firefly luciferase activity.