Fig. 2: SMAD3 and FTO are determined as the direct targets of miR-5581-3p.

a Heatmap assessed from mRNA-seq data listed a part of potential downstream genes of miR-5581-3p. b Volcano plot illustrated enrichment of remarkably differentially expressed genes in miR-5581-3p overexpressed vs. control UM-UC3 cells. c KEGG pathways enrichment from mRNA-seq data. d Venn diagram illustrated the intersected genes from mRNA-seq and online databases including miRWalk, TargetScan, and ENCORI. e The relative content of SMAD3 and FTO in BCa were associated with miR-5581-3p in ENCORI database. f A significant downregulation of SMAD3 and FTO were detected via qRT-PCR assay in UM-UC3 and T24 cell lines post over-expression of miR-5581-3p. g Dual-luciferase enzyme reporter assay illustrated that miR-5581-3p remarkably reduced the luciferase enzyme activity of vectors harboring the 3′-UTRs of SMAD3 and FTO. h Schematic diagram illustrating the miR-5581-3p-targeting sites of SMAD3 and FTO with seed matching. i Western blot data demonstrating decreased protein concentration of SMAD3, FTO, and other proteins in cells transfected with miR-5581-3p mimics. j Western blot data demonstrated the protein levels of SMAD3 and FTO changed in BCa cells transfected of miR-5581-3p mimics at the concentration of 0, 25, 50, and 75 nM. Error bars designate SD acquired from three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.