Luciferase reporter assay

The TLR4 3′UTR segments containing the miR-140 binding sites were amplified using PCR. To amplify TLR3 3′UTR, the genome DNA from A549 cells were extracted using an EasyPure Genomic DNA kit (cat. no. EE101-02, Beijing TransGen Biotech Co., Ltd., Beijing, China). PCR was performed using a 2×EasyTaq PCR SuperMix kit (cat. no. AS111-02; Beijing TransGen Biotech Co., Ltd.). The DNA polymerase used was part of this kit. 2 µl genome DNA (2 µl), 1 µl forward primer, 1 µl reverse primer, 25 µl 2×EasyTaq PCR SuperMix, and 20 µl nuclease-free water were mixed. The thermocycling conditions were as follows: 94°C for 5 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, and then 72°C 10 min. The primers used were as follows: TLR4-3′UTR forward, 5′-GGCTCCTGATGCAAGATGCCCCT-3′ and reverse, 5′-CTGCCTTGAATACCTTCACACGT-3′; GAPDH forward, 5′-CTGAGCACCAGGTGGTCTC-3′ and GAPDH reverse, 5′-CATGACAAGGTGCGGCTCC-3′. Oligonucleotides were then inserted into a pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corp., Madison, WI, USA) and sequenced to confirm there were no mutations. 293 cells were seeded in 24-well plates and co-transfected with miR-140 mimics or negative control miR with recombinant pmirGLO. The relative Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corp.) following 48 h co-transfection.