Fig. 6

Nectin-4 activates PI3K/AKT/NF-κB signaling through modulating miR-520c-3p. A Schematic illustration of the complementary sequence between miR-520c-3p and AKT1(or P65). B Luciferase reporter vectors containing Wt or Mt AKT1 and P65 3′-UTR were constructed and co-transfected with miR-520c-3p mimics, or NC mimics into 293 T cells. Luciferase reporter assays used to determine whether miR-520c-3p directly binds to the 3′-UTR of AKT1 or P65. C, D The effects of miR-520c-3p silencing or overexpression on the expression of AKT, p-AKT, P65, and p-P65 in U2OS and143B cell lines by RT-qPCR and Western blotting, respectively. E The effects of miR-520c-3p silencing or overexpression on the migration ability in U2OS and 143B cell lines by transwell trials, respectively (scale bars 500 μm, magnifications of 100×). F The protein expression levels of EMT-related (ZO-1 and Vimentin) and PI3K/AKT/NF-κB pathway-related markers (AKT, p-AKT, P65, and p-P65) in Nectin-4-OE U2OS cells transfected with miR-520c-3p mimics, and in shNectin-4#2 143B cells transfected with miR-520c-3p inhibitor. G The migration ability of Nectin-4-OE U2OS cells transfected with miR-520c-3p mimics, and shNectin-4#2 143B cells transfected with miR-520c-3p inhibitor (scale bars 500 μm, magnifications of 100×). Each assay was repeated at least three times. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001