Luciferase Reporter Assay for miRNA Binding

To prepare the reporter constructs, the 3′UTR of target genes containing the putative miR-181 binding sites were amplified by PCR and HeLa cDNA as a template. The amplified PCR products were cloned the downstream of the luciferase gene in the pGL3-luc vector (Promega, Madison, WI, USA) as schematically depicted in Figure 6 [12 (link)]. For generation of the mutant reporters, three nucleotide mutations were introduced into the putative miR-181 binding sites using a QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). All primers were purchased from Eurofins (Milan, Italy). HeLa cells were cotransfected with reporter plasmid (200 ng), pRL-CMV-Renilla plasmid (10 ng) and 10 nM miRNA in 96-well plates using Lipofectamine 2000 (Invitrogen, California, CA, USA) according to the manufacturer’s instructions. After 48 h of transfection, luciferase activity was measured using a Dual Luciferase Reporter Assay system (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Firefly luciferase activity was normalized to Renilla luciferase activity.