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. 2017 Jun 20;117(4):525–534. doi: 10.1038/bjc.2017.181

Figure 4.

Figure 4

FOXK1 is a direct target of miR-646. (A) The putative miR-646-binding sites in the FOXK1 3′-UTR; nt, nucleotides. (B) Luciferase activity in the indicated GC cells following transfection of conserved miR-646 binding site-mutated 3′-UTR-driven reporter constructs; wt, wild type; mut, mutant; site 1, the miR-646 motif spanning nt 5062–5068 nt; site 2, the motif spanning nt 5331–5338 nt in the FOXK1 3′-UTR; n=3. **P<0.05 and *P>0.05. (C) Western blot analysis of FOXK1 protein expression in GC cells transfected with miR-646 mimics or inhibitor. Untransfected cells, as well as cells transfected with a control mimic or inhibitor plasmid, were used as controls. (D) In human GC tissues, FOXK1 was negatively correlated with miR-646 at the mRNA level (n=74). (E) The FOXK1, E-cadherin, and vimentin protein levels were detected by western blotting in cells transfected with a FOXK1 expression plasmid, miR-646 mimics, or m-NC. The relative protein expression levels were quantified by comparing the gray level of each band using Quantity One Software (Life Science Research, Hercules, CA, USA). (F) Migratory activity of cells transfected with a FOXK1 expression plasmid and/or miR-646 mimics was evaluated by transwell invasion assay. ****P<0.001. All of these experiments were repeated three times with identical findings.