Fig. 3

FVTF targets inhibiting DNMT1/miR-34a-5p/FoxM1 axis in HCC. A Heatmap of differential expression cancer stem cell-related genes in the FVTF-treated MHCC97H cells and control cells identified by PCR array. Each row represents a mRNA, and each column represents a sample. B Column chart showing 15 up-regulated mRNAs and 4 down-regulated mRNAs in the FVTF-treated MHCC97H cells identified by PCR array. C qRT-PCR showing the relative expression levels of 17 miRNAs in the FVTF-treated MHCC97H cells and control cells. D, E qRT-PCR (D) and Western blot analysis (E) showing the mRNA and protein expression levels of DNMT1 in the HCC cells treated with indicated concentrations of FVTF. F 3′UTR dual luciferase reporter assay. The predicted miR-34a-5p-binding sites in the wild-type (WT) FoxM1 3’ UTR and mutant (Mut) FoxM1 3’ UTR are shown (top); WT or Mut FOXM1 3’ UTR dual luciferase reporter vector was cotransfected into HCC cells with control or miR-34a-5p mimic, and firefly luciferase activity normalized to renilla luciferase activity is shown (bottom). G Western blot showing FoxM1 expression level in the HCC cells transfected with control or miR-34a-5p mimic. H Bisulfite pyrosequencing assay showing the methylation levels of miR-34a-5p promoter CG sites in the FVTF-treated HCC and DNMT1 OE cells. The representative results (top) and statistical analysis (bottom) of miR-34a-5p promoter methylation levels are shown. I Immunohistochemistry showing the expression levels of DNMT1 and FoxM1 in the xenografts with or without FVTF treatment. J qRT-PCR showing the expression levels of miR-34a-5p in the xenografts with or without FVTF treatment. Scale bars = 50 μm. ^*P < 0.05; ^**P < 0.01; ^**P < 0.001; ^****P < 0.0001; ns, no significance.