Validation of the targeting effect of microRNAs on Sox9. (A) The diagram illustrates the construction of Sox9 3' UTR luciferase reporter. (B) The conserved sequences of miR-101 and the 3'UTRs of Sox9 in different species are compared. The underlined sequences indicate a sequence complementary in miR-101 to a specific binding site within the 3'UTR of Sox9. The sequences in the frame were used in our study. (C) Luciferase activity of the Sox9 3'UTR reporter was analyzed in HeLa cells. miRNAs were co-transfected with the wild-type Sox9 3' UTR or mutant vector. Scrambled 22 nt mimic (miR-Scr) was used as a negative control; n = 3,*P < 0.05 versus miR-Scr group. MT refers to mutant Sox9 3'UTR reporter. Primary rat chondrocytes were transfected with the miRNA mimic and miRNA inhibitor. miR-Scr was used as a negative control. Sox9 expression was analyzed by real-time PCR and western blot 24 h post miRNA transfection (D and E). Lower panels (E) show the densitometric analysis of Sox9 expression (upper panels), which is normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The analysis was performed using images from three independent experiments; n = 3,*P < 0.05 versus miR-Scr group.