Fig. 4

miR-196a-5p endocompetes with SMAD6 acting on BMP pathway to influence EMT process. a &d The overexpression and downregulation of miR-196a-5p was tested in MKN45 and MKN74, respectively (Data was expressed as mean ± SD (n = 3) * = significant, * P < 0.05; **, P < 0.01,***P < 0.005; **** < 0.001, Student t test); b) &c) &e) &f) After overexpressed and down regulated miR-196a-5p, the mRNA level of SMAD6 changes negtively with miR-196a-5p; while the mRNA level of BMP4 changes positively with miR-196a-5p. (Data was expressed as mean ± SD (n = 3) * = significant, * P < 0.05; **, P < 0.01,***P < 0.005; **** < 0.001, Student t test); g Western blot for down and up regulated miR-196a-5p in MKN45 and MKN74 to test protein level of BMP pathway markers, BMP-4, t-smad1/5/8, p-smad1/5/8 and smad6. The figure shown that the level of BMP4 and p-smad1/5/8 changes positively with the level of miR-196a-5p. The ratio of p-smad1/5/8 and t-smad1/5/8 indicated that miR-196a-5p influenced activation of BMP pathway. The original blots were shown in Fig. S5A &S5B. h Dual-luciferase report assay. Wild-type and mutated 3’UTR-binding site was cloned in luciferase-reported plasmid and mimcs of miR-196a-5p or nc of miR-196a-5p was transferred with these plasmid into HEK293 cells. Compared with other groups, PGL3-SMAD6WT+ micro-up group normalize luciferase activity decrease by about 40%. The significant reduce prove that the level of SMAD6 decrease when co-transfected with miR-196a-5p. (Data was expressed as mean ± SD (n = 6) * = significant, * P < 0.05; **, P < 0.01,***P < 0.005; **** < 0.001, Student t test). i Effect of BMP pathway inhibitor LDN-193189. Western blot for EMT markers, E-cadherin, N-cadherin, Vimentin, and BMP pathway factors in MKN-45 and MKN-74 respectively as the following groups: micro-up, microup+LDN, microNC+DMSO and micro NC+ LDN. The original blots were shown in Fig. S6.