Easy load pcr master mix

The 3′-UTR of decorin with the predicted miR-181b-5p binding site was amplified by PCR from genomic DNA, and inserted into the pMIR-REPORT^™ luciferase reporter vector (Thermo Fisher Scientific, Inc.) to obtain the wild-type luciferase reporter plasmid (p-decorin-wt). Several nucleotides were mutated in the predicted binding region of miR-181b-5p using PCR, which used the Easy-Load^™ PCR Master Mix (Beyotime Institute of Biotechnology); the thermo profile was as follows: Initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 1 min, and final elongation at 72°C for 1 min. This fragment was cloned into the pMIR-REPORT luciferase reporter vector to generate a mutant reporter plasmid (p-decorin-mut). All constructed plasmids were verified through DNA sequencing. 293T cells were cultured in 96-well plates and co-transfected with miR-181b-5p or NC, and luciferase reporter plasmid using Lipofectamine 3000. After 24 h, the cells were lysed and their luciferase activities were assayed using Dual-Luciferase^® Reporter Assay System (Promega Corporation, Madison, WI, USA). Values were normalized to Renilla luciferase activity.