Dual-luciferase gene reporter assay

The binding site between miR-135a-5p and BAG3 3′UTR was predicted by bio information. A fragment with the binding site for miR-135a-5p of BAG3 mRNA 3′UTR was amplified by PCR and followingly inserted into a pMIR-REPORT luciferase vector (Ambion, Frederick, MD, USA) to construct a BAG3 Wild-Type plasmid (WT-BAG3). Then, partial nucleotides at the binding site were mutated by Gene Mapping Techniques to construct a mutated reporter plasmid (MUT-BAG3). HEK-293 cells were co-transfected with the reporter plasmids and miR-mimics (or miR-inhibitors) or miR-NC (or inh-NC) to verify the binding relationship between miR-135a-5p and BAG3 mRNA 3′UTR. 36 h later, according to the manufacturer's instructions of the luciferase activity detection kit (Ambion, Frederick, MD, USA), the relative luciferase activity of the cells in each group was detected, expressed as the ratio of the firefly luciferase activity to the Renilla luciferase activity.