Fig. 4

IGF2 and FOXM1 are direct targets of miR-4521 in GC cells. a A Venn diagram depicting the overlap of target genes predicted by three miRNA databases (miRTarBase, miRWalk and DIANA-TarBase). b GSEA results were plotted to visualize the correlation between the expression of miR-4521 and genes related to the IGF pathway. c The 3′-UTRs of IGF2 and FOXM1 had potential miR-4521 binding sites. Luciferase reporter vectors containing WT or mutant IGF2 and FOXM1 3′-UTR were constructed and co-transfected with miR-4521 mimics or inhibitors into HEK293T cells. Luciferase reporter assays were used to determine whether miR-4521 directly binds to the 3′-UTR of FOXM1 or IGF2. d RT-qPCR analysis of IGF2 and FOXM1 mRNA expression in SGC7901-H cells with miR-4521 overexpression, SGC7901-L cells with miR-4521 knockdown, and the indicated cells exposed to 1% O₂ for 36 h. e ELISA analysis of IGF2 protein levels in conditioned medium from the indicated cell lines. f Western blot analysis of FOXM1 protein levels in SGC7901-H cells stably expressing miR-4521, SGC7901-L cells stably silencing miR-4521 and lowly invasive cells under hypoxic conditions. g SGC7901-L cells with miR-4521 knockdown were transfected with FOXM1 shRNA or treated with the IGF1R inhibitor PPP (10 nM) for 48 h, while SGC7901-H cells with miR-4521 overexpression were transfected with a plasmid encoding FOXM1 or treated with 100 ng/ml IGF2 for 48 h. Cell invasion was measured by transwell assay. Scale bar, 150 μm. In all cases, error bars denote SD of triplicates. *P < 0.05; ** P < 0.01; ***P < 0.001