Figure 5. DKK1 mRNA is a direct target of miR-376a in OS cells. (A) MiR-376a and its wild-type binding site in the 3′-UTR of DKK1 mRNA. The mutations were introduced into the site complementary to the seed region of miR-376a. (B) The luciferase reporter assay was performed to test whether the 3′-UTR of DKK1 mRNA could be directly targeted by miR-376a in OS cells. HOS and MG-63 cells were cotransfected with either agomir-376a or agomir-NC and either the DKK1-wt or DKK1-mut plasmid. After 48 h of cultivation, the transfected cells were assayed with the Dual-Luciferase Reporter Assay System to measure the luciferase activity. *P < 0.05 vs. the agomir-NC group. (C, D) Expression levels of DKK1 mRNA and protein in miR-376a-overexpressing HOS and MG-63 cells were respectively determined by RT-qPCR and western blotting. *P < 0.05 vs. the agomir-NC group. (E) RT-qPCR was carried out to measure DKK1 mRNA expression in the 47 pairs of OS tissue samples and adjacent-normal-bone tissue samples. *P < 0.05 vs. the normal bone tissues. (F) Spearman’s correlation analysis confirmed the negative correlation between DKK1 mRNA and miR-376a levels among the OS tissues. r = -0.6236, P < 0.0001.