Fig. 3

BRD4 and KPNA4 acted as two targets of miR-596 in EOC. a As indicated in the heatmap, qRT-PCR analysis unveiled the expression pattern of possible miR-596 targets in EOC cells relative to HOSEpiC cells. b The outcomes of qRT-PCR proved that only BRD4 and KPNA4 were targeted by miR-596 in both A2780 and OVCAR3 cells. c Heightened protein levels of BRD4 in EOC cells was disclosed by western blot. d, e The results of qRT-PCR and western blot testified the enhanced or reduced mRNA and protein levels of BRD4 in A2780 and OVCAR3 cells responding to miR-596 inhibitor or miR-596 mimics. f The sequences of BRD4 3′UTR with wild-type or mutant miR-596 binding sites were designed for dual-luciferase reporter assays. g As examined by luciferase reporter assay, the luciferase activity of BRD4 (WT) was declined whereas that of BRD4 (Mut) was not affected in EOC cells with miR-596 upregulation. h The high enrichment of BRD4 in the complex pulled down by biotinylated miR-596 sense was examined by RNA pull-down assay. i Elevated protein levels of KPNA4 in EOC cells was validated by western blot. j, k The outcomes of RT-PCR and western blot uncovered that KPNA4 expression in EOC cells was enhanced by miR-596 inhibitor and decreased by miR-596 mimics. l The sequences of KPNA4 3′UTR containing wild-type or mutant miR-596 binding sites were designed for dual-luciferase reporter assays. m Luciferase reporter assay results indicated that miR-596 interacted with KPNA4 at the predicted binding sites. n The high enrichment of KPNA4 in the complex captured by biotinylated miR-596 sense was validated by RNA pull down assay plus qRT-PCR analysis. *P < 0.05 and **P < 0.01