Fig 2: FOXM1 directly binds to the KIF4A promoter and activates gene transcription. a Map of FOXM1 binding site sequence. b Schematic illustration of four potential FOXM1 binding sites (BS1, BS2, BS3, and BS4) in the KIF4A promoter (top). The 9-base pair sequence of the FOXM1 consensus site and sequences of four FOXM1-binding sites in the KIF4A promoter are shown. Chromatin was isolated from HepG2 cells, and ChIP assay was performed with goat IgG control and FOXM1-specific antibodies (bottom). c Full-length KIF4A luciferase construct was transfected into HepG2 cells with empty vector or the FOXM1 overexpression plasmid for 48 h. Transcriptional activation was analyzed with the dual luciferase reporter assay, with human CCNB1-luciferase construct used as a positive control. d Luciferase constructs for wild-type, or mutant KIF4A promoter was transfected into HepG2 cells along with FOXM1 overexpression plasmid or control vector for 48 h. Transcriptional activation was analyzed with the dual luciferase reporter assay. Results were normalized to Renilla luciferase activity and are expressed as fold change in luciferase activity relative to the control. Data represent the mean ± SD of three independent experiments. **p < 0.01

Fig 3: FOXM1 regulates KIF4A expression. a, b HEK293T cells were transfected with overexpression plasmids for specific isoforms of FOXM1 for 48 h and then harvested for western blotting with indicated antibodies (a) and quantitative real-time PCR (b). c, d HepG2, Huh7, and Sk-Hep1 cells were transfected with pcDNA 3.1-Myc/HisA-vector or pcDNA 3.1-Myc/HisA-FOXM1c plasmids for 48 h and then harvested for western blotting with indicated antibodies (c) and quantitative real-time PCR (d). e HEK293T cells transfected with shRNA-Control or shRNA-FOXM1 constructs for 48 h were harvested for western blotting with indicated antibodies. f, g Huh7, and Hep3B cells were transfected with sh-Control or sh-FOXM1 for 48 h and then harvested for western blotting (f) with indicated antibodies and quantitative real-time PCR (g). CENPF and CCNB1 were used as a positive control. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01

Fig 4: KIF4A promotes proliferation and clonogenicity of HCC cellsa The effect of KIF4A knockdown with siRNAs was verified by western blotting 72 h after transfection. b The effect of KIF4A overexpression was verified by western blotting. c Viability of KIF4A knockdown cells was assessed with an MTT assay at the indicated times. d Viability of KIF4A overexpression cells was assessed with an MTT assay at the indicated times. e Colony formation assays of SMMC-7721 and BEL-7404 cells transfected with negative control and KIF4A-targeted siRNAs. Upper panel: representative image, lower panel: quantification of the colony numbers. f Colony formation assays of control and KIF4A-overexpressing HCC cells. Upper panel: representative image, lower panel: quantification of the colony numbers. Statistically significant difference: *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: FOXM1–KIF4A axis promotes HCC progression in vivo. a Growth curves of xenograft tumors formed by indicated Huh7 cells in nude mice. Data represent mean ± SD (n = 8). **p < 0.01. b Image of xenograft tumors. c Weight of xenograft tumors. Data represent mean ± SD (n = 8). **p < 0.01. d Downregulation of KIF4A protein in Huh7 FOXM1 + sh-KIF4A compared to Huh7 FOXM1 + sh-Control xenografts, as confirmed by western blotting. e HE staining of tumor tissue samples from indicated groups and detection of FOXM1, KIF4A, and Ki67 protein levels by immunohistochemistry. f Model for FOXM1-KIF4A axis mediated HCC proliferation. FOXM1 transcriptionally activates KIF4A via directly binding to the promoter of KIF4A, promoting mitosis of cell cycle and proliferation of HCC cells. The inhibition of KIF4A is shown to suppress FOXM1-induced HCC proliferation and growth in this study