Firefly Luciferase Reporter Assay for miR-140-5p Targets

The plasmids used for firefly luciferase reporter assay were packaged by Genechem (Shanghai Genechem, China). And the plasmids designated IGF1R-WT, TGFBRI-WT (wild-type of miR-140-5p, targeting to IGF1R 3′-UTR and TGFBRI 3′-UTR), IGF1R-MU and TGFBRI-MU (mutated miR-140-5p, targeting to IGF1R 3′-UTR and TGFBRI 3′-UTR) were used. The mimic and the mimic negative control of miR-140-5p were purchased from Ribobio (Guangzhou RiboBio, Guangzhou, China). HEK293 cells were cultured in complete medium for 24 h before transfection. 0.05 μg firefly luciferase reporter, 0.05 μg IGF1R/TGFBRI plasmid, and 0.01 μg Renilla luciferase control vector were co-transfected into HEK293 cells by lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. At 48 h post-transfection, luciferase activity was detected by using the Dual-Glo luciferase reporter system (Promega Corp., Madison, WI, USA) in accordance with the protocols of manufacturer [20 (link)]. The relative luciferase activity value was achieved against the renilla control.