Fig. 4. Smurf1 was up-regulated in GC tissues and cells and was confirmed to be a direct target gene of miR-1254.

a Ninety pairs of GC tissues and adjacent normal tissues were collected to examine the relative expression of Smurf1 by qRT-PCR. b Western blot was used to determine Smurf1 protein expression in eight pairs GC specimens and adjacent normal tissues. c Immunohistochemistry staining was used to determine the protein level of Smurf1 in GC tissues and adjacent tissues (scale bar: 50 μm). d The correlation between the expression of miR-1254 and Smurf1. e and f The expression of Smurf1 in GC cells and GES-1 were detected by qRT-PCR and western blot, respectively. g Luciferase reporter assay was conducted to confirm that miR-1254 is directly bound to the 3′-UTR region of Smurf1. The result of Luciferase activity was analyzed in cells co-transfected with miR-1254-mimics or negative control with pGL3-Smurf1-WT or pGL3-Smurf1-MUT. h The expression level of Smurf1 protein in GC cells was verified by western blot. i The expression level of Smurf1 mRNA in GC cells was detected by qRT-PCR. *p < 0.05; **p < 0.01; ***p < 0.001. The data expressed as the mean ± SD