2.6. Dual-Luciferase Assay
LYSED, LYSIS
The sequence of MAP3K1 3′-UTR and 3′-UTR-mutant regions was synthesized and cloned into the pmirGLO Dual-Luciferase miRNA vector (Promega, Madison, USA) by GenScript Company (Nanjing, China). HEK293T cells were cotransfected with the luciferase reporter constructed (500 ng) together with miRNA mimics or negative control (NC, 40 nM) using Lipofectamine 3000. After 48 hr, the cells were washed and lysed in the passive lysis buffer (Promega, Madison, USA). Firefly luciferase (f-luc) and renilla luciferase (r-luc) activities were detected by a dual-luciferase reporter assay system (Promega, Madison, USA). Relative reporter activity was normalized to the r-luc activity.