Plasmid construction and luciferase reporter assay

The 3′-untranslated region (UTR) of human MET and PDL1 were amplified from human genomic DNA and individually inserted into the pmirGLO Dual-Luciferase miRNA Target Expression Vector. Mutant PDL1 and MET 3′-UTRs were generated with the QuickchangeXL mutagenesis kit (Stratagene, United States) to disrupt the binding of miR-34a. The fragment of MET and PDL1 3′-UTR mutant was inserted into the pmirGLO Dual-Luciferase miR Target Expression Vector. CAL27 cells were co-transfected with wild-type or mutant MET reporter plasmid and miR-34a-5p mimic or negative control using Lipofectamine 2000 (Invitrogen). Similar approach was taken to verify interaction of miR-34a-5p with PDL1 3’UTR in RAW macrophages. Luciferase activity was measured forty-eight hours post-transfection using Dual-Glo Luciferase Reporter System according to the manufacturer’s instructions (Promega) using a LB96V luminometer (Berthold). Firefly luciferase units were normalized against Renilla luciferase units to control for transfection efficiency. Luciferase activity was averaged from at least 5 replicates.