Figure 4. ROCK1 is a direct target of miR-421 in PTC cells. (A) The predicted miR-421 binding sites in the 3’UTR of ROCK1 and the mutated sequence are shown. (B) Dual luciferase reporter assay shows the relative luciferase activity in TPC-1 cells co-transfected with miR-421 mimic or miR-NC and luciferase reporter plasmid with wild-type ROCK1-3’-UTR (WT-ROCK1) or mutant ROCK1-3’-UTR (MUT-ROCK1). (C) QRT-PCR analysis shows the ROCK1 mRNA levels in miR-NC- and miR-421 mimic-transfected TPC-1 cells. (D) Western blot analysis shows ROCK1 protein levels in miR-NC- and miR-421 mimic-transfected TPC-1 cells. (E) QRT-PCR analysis shows ROCK1 mRNA expression in 54 paired PTC and adjacent normal thyroid tissues. (F) Spearman correlation analysis shows that ROCK1 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.