Pgl3 basic dual luciferase reporter vectors

Luciferase reporter assays were used to validate the direct binding of miR-93 and ZNF322 mRNA 3′UTR fragments. In brief, the wild-type ZNF322 mRNA 3′UTR fragments containing the predicted binding site of miR-93 were amplified from human genomic DNA by PCR using specific primers. The mutant ZNF322 mRNA 3′UTR fragments were constructed by site-directed mutagenesis using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. The wild-type and mutant ZNF322 mRNA 3′UTR fragments were cloned into the pGL3-basic dual luciferase reporter vectors (Promega, Madison, WI, USA), respectively. The luciferase activity of the reporter vectors carrying wild-type or mutant ZNF322 mRNA 3′UTR fragments was analyzed in the presence of miR-93. A dual-luciferase reporter system (BD Bioscience) was used to determine the relative luciferase activity at 48 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.