Fig. 3
EHF promotes cell migration, invasion, and metastasis through the upregulation of KRT16 in OSCC cells. a Plasmid constructs for the KRT16 promoter–driven luciferase reporter assays including wild-type (KRT16–2249 ~ + 5) and three deletion mutants of ETS binding sites depicted by black boxes. b Left, the expression levels of EHF and KRT16 mRNAs in OC-3 pair were measured using qRT-PCR (*P < 0.05, **P < 0.01). Right, the expression levels of EHF protein in C9 pair and OC-3 pair were analyzed through immunoblotting. c Effect of overexpression (left) or knockdown (right) of EHF on endogenous KRT16 mRNA expression in OC-3-IV cells. d The wild-type and mutant KRT16 promoter–driven expressions of luciferase. e OC-3-IV cells were transfected with pcDNA3.1 (control) or EHF plasmid, and the ChIP assay was performed using primers specific for the site-3 region. The PCR product was analysed with agarose gel electrophoresis (Left) or qRT-PCR (Right). Histone 3 (H3) was used as a positive control. f Immunoblotting of EHF protein in CGHNC9 cells transfected with the EHF-siRNAs or NC-siRNA. g Left, the migration and invasion ability was decreased in CGHNC9 cells transfected with EHF-siRNA-3 compared with the control. Right, cell migration and invasion were increased through ectopic expression of EHF in CGHNC9 cells. h Left, the expression levels of EHF and KRT16 protein in OC-3-IV cells were analyzed through immunoblotting. Right, ectopic expression of KRT16 significantly restored the inhibition of migration and invasion due to EHF knockdown. i Lung metastasis of OC-3-IV-M-shEHF stable cells generated by 1 × 106 cells injected into the tail vein of CB17-SCID mice was inhibited by EHF downregulation and was significantly restored by transfection with KRT16. The respective images display H&E staining of the lung metastases (200x) of each treatment group. Histograms in (b), (c), (d), (e), (g), (h) and (i) represent means ±SD from three independent experiments (*P < 0.05, **P < 0.01). The actin was used as an internal control