YAP1 mRNA as a direct target of gga-miR-375 and the effect of the transfection of gga-miR-375 on the EMT marker levels in DF-1 cells. (a) The complementary site for the seed region of gga-miR-375 is indicated after the alignment of the YAP1 3ʹ UTR, gga-miR-375, and MUT 3ʹ UTR. (b) Relative luciferase activity of YAP1 is dependent on gga-miR-375 (** represent P < 0.01). (c) The confirmation that gga-miR-375 was successfully overexpressed after transient transfection in DF-1 cells (** represent P < 0.01). (d) RT-qPCR reveals that the overexpression of gga-miR-375 downregulated the YAP1 mRNA expression at 24 h (** represent P < 0.01). (e) Western blotting reveals that the overexpression of gga-miR-375 downregulated the YAP1 protein expression at 48 h. The levels of YAP1 protein are shown as fold change next to the images after normalized with β-actin (** represent P < 0.01). (f) Western blotting reveals that the overexpression of gga-miR-375 decreased the N-cadherin, Fibronectin, Vimentin, ZEB1 and MMP2 protein expression, but increased the E-cadherin protein expression compared with the mock-transfected cells at 48 h. The levels of N-cadherin, Fibronectin, Vimentin, ZEB1, MMP2 and E-cadherin protein are shown as fold change next to the images after normalized with β-actin (** represent P < 0.01). (g) RT-qPCR revealed that the overexpression of gga-miR-375 decreased the level of N-cadherin (** represent P < 0.01), Fibronectin (** represent P < 0.01), Vimentin (** represent P < 0.01), ZEB1 (** represent P < 0.01), MMP2 (** represent P < 0.01), but increased that of E-cadherin (** represent P < 0.01) transcription compared to the mock-transfected cells at 24 h. The data represent the mean±s.e.m. of three independent experiments.