Figure 3. DLEU1 sponges miR-421 in PTC cells. (A) The diagram shows the predicted miR-421 binding sites in the 3’UTR of DLEU1 and the mutations in the miR-421 binding sites. (B) Dual luciferase reporter assay shows the relative luciferase activity of TPC-1 cells co-transfected with miR-421 mimic or miR-NC plus luciferase reporter plasmid with the wild-type DLEU1 (WT-DLEU1) or mutant DLEU1 (MUT-DLEU1). The miR-421 binding sites are mutated in the mutant DLEU1. (C) QRT-PCR results show DLEU1 levels in the RNA pull down extracts using biotinylated wild-type or mutant miR-421. (D) QRT-PCR analysis shows miR-421 levels in control and DLEU1-silenced TPC-1 cells. (E) QRT-PCR analysis shows DLEU1 levels in control and miR-421 mimic-transfected TPC-1 cells. (F) QRT-PCR analysis shows miR-421 expression in 54 paired PTC and adjacent normal thyroid tissues.(G) QRT-PCR analysis shows the expression of miR-421 in four PTC cell lines (BHP5-16, 8505C, TPC-1, and SW1736) and the human thyroid follicular epithelial cell line, Nthy-ori3-1. (H) Spearman’s correlation analysis shows that DLEU1 expression is inversely related to miR-421 expression in PTC tissues (n=54). Note: The data are represented as the means ± SD of at least three independent experiments. *P< 0.05 and **P< 0.01.