Functional validation of miR-125b binding the putative target sequence in the 3′-UTR of ADAMTS-4 mRNA. (A) Relative expression level of miR-125b in HEK293T cells transfected with pcDNA3.1-pre-miR-125b-1, -2 or negative control (n = 3). (B) Relative expression of miR-125b in 293T cells and normal chondrocytes (n = 3). (C) Luciferase activity in 293T cells co-transfected with pLuc2-ADAMTS-4 3'-UTR or -ADAMTS-4 3'-UTR mutant and pcDNA3.1-pre-miR-125b-1, -2, -125b-1 mutant, -125b-2 mutant or pcDNA3.1 (n = 3). The data are expressed as mean and SEM of three independent experiments, each of which was run in triplicate. ** = P <0.001 as measured using an unpaired Student's t test. ADAMTS-4, aggrecanase-1; pre-miR-125b MiRNA-125b precursor; SEM, standard error of the mean; 3'-UTR, 3'-untranslated region.