Sequencing and clinical data of patients with HCC were obtained from two public cohorts, namely TCGA (http://xena.ucsc.edu) and ICGC (https://dcc.icgc.org). A total of 373 and 243 patients from the TCGA and ICGC JP cohorts, respectively, were included in the analysis. Patients with incomplete OS or disease-free survival (DFS) information were excluded. OS was defined as the time from the date of initial pathological diagnosis to the time of death or last follow-up, while DFS was defined as the time from first treatment to the time of tumor recurrence or death. The TCGA cohort was used as the exploration cohort (clinicopathological information is provided in Table I), while the ICGC JP cohort was used for validation.

The human Huh7 cell line (American Type Culture Collection) was cultured in a growth medium consisting of Dulbecco's Modified Eagle's Medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 4.5 g/l glucose, 10% fetal bovine serum (FBS; Shanghai ExCell Biology, Inc.) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Complementary DNA (cDNA) was synthesized using the ReverTra Ace qPCR RT Master Mix with a gDNA Remover kit (Toyobo Life Science) according to the manufacturer's instructions. qPCR was then performed in a 25-µl volume reaction mixture containing 12.5 µl AceQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.), 2 µl template cDNA (100 ng/µl) and 1 µM of primers in a LightCycler 96 (Roche Diagnostics Co., Ltd.). The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 94°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. GAPDH was used as a normalization control. Each experiment was performed independently at least three times and the fold change in the expression of each gene was calculated using the 2^−ΔΔCq method (40). Primers for qPCR were obtained from BioSune Biotechnology Co., Ltd. All primers were designed to cross an intron-exon junction sequence to minimize genomic DNA contamination. The primer sequences were as follows: qPCR-PLK1-forward (F), 5′-AAGAGATCCCGGAGGTCCTA-3′ and qPCR-PLK1-reverse (R), 5′-GCTGCGGTGAATGGATATTT-3′; qPCR-FOXM1-F, 5′-CGTGGATTGAGGACCACTTT-3′ and qPCR-FOXM1-R, 5′-TCTGCTGTGATTCCAAGTGC-3′; qPCR-GAPDH-F, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and qPCR-GAPDH-R, 5′-GCCATCACGCCACAGTTTC-3′.

Cells were lysed in radioimmunoprecipitation assay cell lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitors (cat. no. HY-K0010; MedChem Express) and phosphatase inhibitor cocktail (cat. no. 78427; Thermo Fisher Scientific, Inc.). Protein concentrations were determined via a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Proteins were separated using 10% SDS-PAGE [gels using 1X running buffer and transferred to polyvinylidene difluoride membranes (MilliporeSigma)]. Afterwards, the membranes were blocked by 5% skimmed milk (Anchor; Fonterra Co-operative Group) at room temperature for 1 h and incubated with primary antibodies against FOXM1 (rabbit; cat. no. A2493; dilution, 1:1,000; Abclonal), PLK1 (rabbit; cat. no. 208G4; dilution, 1:1,000; Cell Signaling Technology, Inc.) and GAPDH (mouse; cat. no. AC002; dilution, 1:10,000; Abclonal) at 4°C overnight. Subsequently, the membranes were cleaned twice with TBS with 0.1% Tween-20 and incubated with secondary goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (dilution, 1:5,000; cat. no. G21234; Thermo Fisher Scientific, Inc.) or goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (dilution, 1:5,000; cat. no. G21040; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Proteins were detected via enhanced chemiluminescence (Vazyme Biotech Co., Ltd.) with a digital luminescent image analyzer (Tanon-4200; Tanon Science and Technology Co., Ltd.). The intensities of protein bands were semi-quantified using ImageJ software (ImageJ bundled with 64-bit Java 1.8.0_172; National Institutes of Health).

Human FOXM1 small interfering RNA (siRNA), human PLK1 siRNA and control siRNA were obtained from Shanghai GenePharma, Co., Ltd. The siRNA oligonucleotides were transfected into Huh7 cells (at 70% confluence) using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. siRNAs were mixed with Opti-MEM reduced serum medium (Gibco; Thermo Fisher Scientific, Inc.) and Lipofectamine RNAiMAX and incubated for 20 min at room temperature. Subsequently, siRNA/Lipofectamine in Opti-MEM was diluted 1:5 (corresponding to a final concentration of 50 nM) in differentiation medium, and added to the cells. Thereafter, cells were incubated for 48 h at 37°C with 5% CO₂. The following siRNAs were used for the experiments, which had the following sequences: siFOXM1-1, 5′-GCUGGGAUCAAGAUUAUUATT-3′; siFOXM1-2, 5′-GGCUGCACUAUCAACAAUATT-3′; siPLK1-1, 5′-CCCUCACAGUCCUCAAUAATT-3′; siPLK1-2, 5′-GGCAACCAAAGUCGAAUAUTT-3′; si negative control (NC), 5′-ACGUGACACGUUCGGAGAATT-3′.

A CCK-8 kit (Vazyme Biotech Co., Ltd.) was used to measure the proliferation of Huh7 cells. A total of 1,000 cells in a volume of 100 µl per well were cultured in six replicate wells in a 96-well plate in medium containing 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO₂ for 6 h. When cells had adhered, CCK-8 reagent (10 µl) was added to 90 µl DMEM to generate the working solution, of which 100 µl was added per well and incubated for 2 h. This assay was performed at 0, 24, 48, 72 and 96 h. The optical densities were measured at a spectral wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.). Six replicates were analyzed for each time point.

To measure the DNA replication activity of the Huh7 cells, a Cell-Light EdU Apollo488 in Vitro Kit (cat. no. C10310-3; Guangzhou RiboBio Co., Ltd.) was used. Huh7 cells were seeded in 96-well plates at a density of 8×10³ cells per well. After 24 h, the cell culture medium was replaced with 50 µM EdU solution diluted with growth culture medium, followed by incubation for 2 h. The cells were then processed using the Cell-Light EdU Apollo488 in Vitro Kit according to the manufacturer's protocol. Images were captured with an Olympus fluorescence microscope (BX53; Olympus Corporation).

A total of 1×10⁶ cells were washed twice with PBS and fixed overnight with 1 ml pre-cooled 75% ethanol at 4°C. Thereafter, cells were collected by centrifugation (500 × g; 5 min; 4°C), washed twice in PBS and incubated with propidium iodide (5 ug/ml, Sigma) and RNase A (0.1 mg/ml; Thermo Fisher Scientific, Inc.) for 30 min at 4°C in the dark. A 40-µm screen filter was then used to filter the cell suspension and remove any adhesive cells. This was followed by flow cytometry to analyze the DNA content using the BD LSRFortessa system (BD Biosciences). FlowJo_v10 software (Tree Star, Inc.) was used to estimate the proportion of cells in the G0/G1, S and G2/M phases.

The pharmacological PLK1 inhibitor BI 2536 (HY-50698) was purchased from MedChemExpress. The drugs were reconstituted in DMSO and aliquots were stored at −20°C. An equivalent amount of DMSO was used for each experiment as a vehicle control. For cell proliferation and cell cycle assays, Huh7 cells were incubated with 10 nM BI 2536 for 24 h.

Statistical analysis was performed using R software (version 4.0.3) for survival analysis and Cox analysis, and GraphPad Prism (version 8.0; GraphPad Software, Inc.) for others. Kaplan-Meier curves were used to estimate OS and DFS. The log-rank test was used to compare patient survival times between high and low gene expression groups and hazard ratios (HRs) were calculated using the Cox proportional hazards model. The combined expression of FOXM1 and PLK1 was dependent on FOXM1 expression and PLK1 expression, thus they were analyzed respectively in Multivariable Cox analysis. Due to the crossing of survival curves, the ‘TSHRC’ package (v0.1-6; http://CRAN.R-project.org/package=TSHRC) of R software, which is a two-stage procedure for comparing HR functions, particularly suited for situations where HR functions cross, was used to perform a two-stage test rather than the log-rank test (41,42). Pearson's correlation coefficient was calculated to analyze the correlation between FOXM1 and PLK1 expression. Differences between 2 groups were analyzed using the unpaired Student's t-test with or without Welch's correction. One-way ANOVA followed by Tukey's post-hoc test was used for comparisons between multiple groups. Values are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The TCGA cohort was used as the exploration cohort and the ICGC JP cohort was used for validation. The baseline characteristics of the TCGA data set analyzed in the present study are summarized in Table I. The mean age of TCGA cohort was 59.5 years and the percentage of men was 67.6%. The most prevalent hepatitis virus was HBV. The ICGC JP cohort contained sequencing and clinical data of 243 patients with HCC from Japan. The mean age of the ICGC cohort was 67.5 years and the percentage of men was 74.9%. Most patients presented with primary tumors (98.8%) and approximately half of the patients had stage II tumors (45.3%).

The expression of FOXM1 and PLK1 in HCC and their effect on survival were first analyzed (Fig. 1). Compared with non-tumor liver tissues, the tumor tissues exhibited significantly higher FOXM1 expression in the TCGA (P<0.001; Fig. 1D) and ICGC JP (P<0.001; Fig. 1E) cohorts. To investigate the prognostic value of FOXM1 and PLK1 expression in HCC, the patients with HCC were divided into low and high expression groups at the 50th percentile. In addition, in the TCGA cohort, patients with high FOXM1 expression levels (F^H) had shorter OS [HR, 1.68; 95% confidence interval (CI), 1.18-2.38; P=0.003; Fig. 1A] and DFS (HR, 1.70; 95% CI, 1.26-2.30; P<0.001; Fig. 1C). Consistently, F^H was associated with poor prognosis in the ICGC JP cohort (HR, 4.08; 95% CI, 2.05-8.12; P<0.001; Fig. 1B).

As presented in Fig. 2, similar to FOXM1, PLK1 was highly expressed in HCC tumor tissues in both the TCGA (P<0.001; Fig. 2D) and ICGC JP (P<0.001; Fig. 2E) cohorts. In addition, in the TCGA cohort, patients with high PLK1 expression level (P^H) had shorter OS (HR, 2.05; 95% CI, 1.43-2.93; P<0.001; Fig. 2A) and DFS (HR, 1.57; 95% CI, 1.16-2.11; P=0.003; Fig. 2C). In the ICGC JP cohort, patients with P^H also had significantly poorer OS (HR, 3.83; 95% CI, 1.93-7.61; P<0.001; Fig. 2B).

To assess the independent predictive value of F^H and P^H, logistic regression with a multivariate Cox proportional hazards model was utilized. After adjusting for age, sex, stage and virus status, stage and virus status were identified as independent prognostic factors for OS. However, neither F^H (HR, 1.14; 95% CI, 0.67-1.95; P=0.634; Table II), nor P^H (HR, 1.73; 95% CI, 1.00-2.99; P=0.052; Table II) were significantly and independently associated with a shorter OS.

FOXM1 and PLK1 are essential for cell cycle progression. PLK1 itself is a target of FOXM1 and phosphorylation of FOXM1 mediated by PLK1 is required for FOXM1 transcription activation (14,16). In both the TCGA (r²=0.793, P<0.001; Fig. 3A) and ICGC JP (r²=0.714, P<0.001; Fig. 3B) cohorts, a positive linear correlation between FOXM1 and PLK1 expression was observed, consistent with the presence of a feedback loop between them in HCC tissues. In the TCGA cohort, patients with combined high expression of FOXM1 and PLK1 (F^H-P^H) exhibited significantly shorter OS (HR, 2.04; 95% CI, 1.40-2.97; P<0.001; Fig. 3C) and DFS (HR, 1.73; 95% CI, 1.26-2.38, P<0.001; Fig. 3E). The median OS corresponding to the F^H-P^H (33.02; Fig. 3C) group was shorter than that corresponding to the F^H (45.07; Fig. 1A) and P^H (37.75; Fig. 2A) groups. These observations were validated in the ICGC JP cohort (Fig. 3B). Furthermore, the prognostic value of F^H-P^H expression in patients with HCC was confirmed using logistic regression with a multivariate Cox proportional hazards model. The results indicated that F^H-P^H expression was the most significant independent predictor of OS (HR, 1.94; 95% CI, 1.31-2.89; P=0.001; Table III).

Cancer immunotherapy has revolutionized cancer treatment. Antibodies against programmed death 1/ligand 1 and cytotoxic T-lymphocyte-associated protein 4 are effective for the treatment of HCC (43–45). Given that the immune microenvironment has an important role in the response to immunotherapy (46,47), the impact of F^H-P^H expression on the immune microenvironment in patients with HCC was evaluated. Low activation of the transforming growth factor (TGF)-β pathway is associated with better clinical outcomes for patients with cancer (32). Thus, the TGF-β response score (48) were applied in patients with HCC and it was observed that the F^H-P^H group had a relatively higher score (P<0.001; Fig. 3F). Furthermore, CIBERSORT (49) has been used to analyze immune cell infiltration. Its application in the present study suggested that regulatory T (Treg) cells were significantly enriched in the F^H-P^H group (P<0.001; Fig. 3G). Of note, Treg cells are able to inhibit T-cell proliferation and cytokine production and have a critical role in preventing tumor immune response (50,51). These results suggested that F^H-P^H expression is associated with a suppressive immune microenvironment and may hamper immunotherapy treatment in HCC.

Given that the overexpression of FOXM1 and PLK1 was associated with poor outcomes of HCC and based on their key regulatory roles in cell cycle progression, it was investigated whether FOXM1 and PLK1 influence HCC progression (Figs. 4 and 5). siRNA targeting FOXM1 and PLK1 was synthesized and the knockdown efficiency was measured in Huh7 cells (Figs. 4A and B and 5A and B). The cell proliferation rate was estimated using CCK-8 and EdU assays. FOXM1 knockdown decreased Huh7 cell viability (Fig. 4C), as well as the percentage of EdU-positive Huh7 cells (Fig. 4D), indicating a lower proportion of cells entering the DNA replication phase of the cell cycle. PLK1 knockdown exerted a similar effect on Huh7 cells (Fig. 5C and E). To confirm the requirement of PLK1 for Huh7 cell proliferation, a pharmacological inhibitor of PLK1, BI 2536 (38), was applied during the CCK-8 assay. In the presence of the inhibitor, a significant decrease in the proliferation of Huh7 cells was observed (Fig. 5D). Furthermore, flow cytometry suggested that knockdown of FOXM1 (Fig. 4E) or PLK1 (Fig. 5F) resulted in a marked increase in the proportion of cells in S and G2/M phase, as well as a decrease in the proportion of cells in G1 phase at 24 h after transfection. It appears that, as more cells progress away from G0/G1 phase, the cells are trying to proliferate but they exhibit cell cycle arrest in S and G2/M phase. Consistent with this observation, Huh7 cells treated with BI 2536 also exhibited a marked increase in the proportion of cells in S and G2/M phase (Fig. 5G). These results suggested that FOXM1 and PLK1 are required for HCC cell proliferation. Insufficient FOXM1 or PLK1 were thus indicated to hamper cell cycle progression as well as the proliferation of HCC cells, providing additional evidence that the coordinated overexpression of FOXM1 and PLK1 is an independent prognostic factor for HCC.