Pmir report vector kit

Wild type or mutated 3′-UTR of PDRG1 mRNA were clone to the downstream of the luciferase reporter open reading frame, and the promoter region of miR-519d containing two HRE (HRE-wt) or the mutated version (HRE-mut) were cloned to the upstream promoter region of pGL3-enhancer plasmid using the pMir-Report vector kit (Applied Biosystems, Carlsbad, CA, USA). 10⁵/well cells transduced with indicated plasmid were seeded in 24 well plate and transfected with indicated luciferase constructs by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were allowed to grow 24 h after the transfection. The luciferase activities were determined with a dual-luciferase reporter assay kit (Promega, Madison, WI, USA) according to the instructions.

Wild‐type (LRP6‐wtUTR) and mutated (LRP6‐mutUTR) miR‐590‐3p targeting sequences from LRP6 3′‐UTR were cloned into the downstream of a luciferase reporter gene on the pGL3‐enhancer plasmid using the pMir‐Report vector kit (Applied Biosystems, Carlsbad, CA, USA). Cells at the density of 10⁵ per well were plated in 24‐well plate and transfected with indicated luciferase constructs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), then allowed 24 hr to grow after the transfection before various experiments. The activities of luciferase were determined using a dual‐luciferase reporter assay kit (Promega, Madison, WI, USA) following the manufacturer's instructions.