Fig. 2

IRS1 is a direct target gene regulated by miR-203. a Schematic representation of IRS-1 3′-UTR showing putative miR-203 target sites, which were conserved across species. b Luciferase activity assay was performed with IRS-1 3′-UTR construct or control construct co-transfected with AD-miR203 (Firefly luciferase values were normalized to Renilla luciferase activity), which was significantly decreased when either IRS-1-site A or IRS-1-site B was present in the constructs, whereas mutation or deletion of the seed sequences (IRS1-Mut A, Del A and IRS1-Mut B, Del B) restored reporter gene activity. Expression of miR-203 alone had no effect on reporter gene activity when no seed sequences were inserted. c Relative expression of miR-203 in DU145 and PC-3 cells infected with miR-203 virus was determined by Q-PCR. Data represent mean ± SD with three replicates, *** P < 0.0005. d Relative expression of IRS-1 in DU145 and PC-3 cells infected with miR-203 virus was determined by Q-PCR. Data represent mean ± SD with three replicates, ns: not significant. e Protein expression of IRS-1 in DU145 and PC-3 cells was determined by Western blot after miR203-transfection. β-tubulin serves as internal control. IRS-1 levels were quantified with Image J software and normalized to the internal control. The full-length blots of panel 2e and 3b were presented in Supplementary Figure 1