fig2

Figure 2. TWIST1 is a miR-186-5p target. Inset: The putative binding site of miR-186-5p within the Twist1 3’-UTR is shown, with the paired sequences of the wt and mutant (mut) constructs generated. (A) The pMIR-Twist1-WT and pMIR-Twist1-MT vectors were transfected into the three MPM cell lines and in HEK293 cells along with miR-186-5p or ctrl mimics and luciferase activity was assessed 48 h later. The percentage of luciferase activity in the cells transfected with miR-186-5p mimics over the cells transfected with the ctrl-mimics is reported. Means ± SD of three replicates are shown. *P < 0.05, **P < 0.01. (B) Twist1 protein levels in NCI-H2373 cells treated with ctrl or miR-186-5p mimics or butein and ctrl (DMSO 0.01 %) as assessed by Western blotting. Anti-tubulin staining was used as a loading control. Asterisk (*) indicates the TWIST1-specific protein band. (C) Histograms reporting the relative (normalized) protein band intensity of TWIST1 from all three MPM cell lines treated as indicated in (B). The average values of two independent experiments are reported. **P < 0.01 (vs. ctrl).