Double luciferase report system detection kit

The MicroRNA.org website (http://www.microrna.org/microrna/getMirnaForm.do) was used to predict the target gene of miR-9, and the dual luciferase reporter assay was used to confirm the findings. Luciferase activity was detected using the double luciferase report system detection kit (Promega Corp., Madison, WI, USA). Firstly, hsa-miR-9 mimics and miRNA negative control were co-transfected with p16-3′UTR or p16-3′UTR mutant plasmids (400 ng) into 293 cells using Lipofectamine™ 2000 for 48 h. The cells were then lysed using 1X passive lysis buffer (PLB) at 37°C for 15 min. The cell suspension was transferred to a black enzyme plate. LARII and stop&Glo^® reagent (Promega Corp.) was then added to the plate in a drop-wise manner. Luciferase activity was measured using a GloMax^® Discover Multimode Microplate Reader (GM3000; Promega Corp.). pRL-TK (Promega Corp.) was used as an internal control.

Targetscan 7.2 (http://http://www.targetscan.org/.) predicted that CREB was the potential target for miR-450a-5p. For determination, double-luciferase report system detection kit (Promega, Madison, Wisconsin, USA) was used for double-luciferase reporter analysis. Briefly, HUVECs were seeded in 24-well plates (1×10⁵ cell/well) for 24 h. Then, miR-450a-5p mimic or blank control was transfected into HUVECs combined with wild type CREB (CREB-WT) or its mutant type (CREB-MUT) using Lipofectamine^TM 2000. After 48 h, the cells were lysed and the luciferase activity was evaluated with the GloMax^® Discover Multimode Microplate Reader (GM3000, Promega, USA).