2.7. Luciferase activity assay

To generate reporter vectors bearing miRNA-binding sites, the 3′-untranslated region (3′-UTR) of PTEN and its mutant variant were synthesized by Sangon (Shanghai, China). The construct was inserted into the multiple cloning sites downstream of the luciferase gene (Hind III and Sac I sites) in the pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, USA). For luciferase assay, 0.1 μg of luciferase reporters containing 3′-UTR was co-transfected with NC, miR-499a, or AMO-499a and 10 ng of PRL-TK (TK-driven Renilla luciferase expression vector) into HEK-293 cells using X-treme gene siRNA transfection reagent (Roche, Switzerland), according to the manufacturer's instructions. Luciferase activity was measured 48 h after transfection with a dual luciferase reporter assay kit (Promega, USA).