Pmir rb luciferase reporter vector

A SOD1 3′-UTR sequence containing an miR-409-3p binding site was cloned and inserted into the pmiR-RB-luciferase reporter vector (Promega, United States), to generate the SOD1 3′-UTR-WT construct. Mutation of the SOD1 miR-409-3p binding site was conducted by site-directed mutagenesis using the Quick Change Lightning kit (Stratagene, La Jolla, CA, United States) to generate the SOD1 3′-UTR-MUT construct. For the experiments: cells were plated in 24-well plates, incubated overnight, and then H460 cells co-transfected with SOD1 3′-UTR-WT, SOD1 3′-UTR-MUT, miR-409-3p mimic, or miR-409-3p inhibitor. Luciferase activity was tested using the Dual-Luciferase Reporter Assay System (Promega, United States). Renilla activity was used to normalized luciferase activity values for each sample.