From 2006 to 2008, 75 patients admitted to Shanxi Dayi Hospital were enrolled in the present study. In each case, HCC and adjacent non-tumorous tissues were snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction. Follow-up was completed in October 2013. Ethical approval for the present study was provided by the Independent Ethics Committee of Shanxi Dayi Hospital. Informed and written consent was obtained from all patients or their advisers according to the ethics committee guidelines.

HepG2, BEL-7404, SMC-7721, MHCC-97L and MHCC-97H cells were obtained from the Cell Bank of Shanghai Biology Institute, Chinese Academy of Science (Shanghai, China). All culture media (Life Technologies, Carlsbad, CA, USA) were supplemented with 10% fetal bovine serum (FBS; Life Technologies), 100 mg/ml penicillin G and 50 µg/ml streptomycin (Life Technologies). HepG2, BEL-7404, MHCC-97L and MHCC-97H cells were cultured in DMEM. SMC-7721 cells were cultured in RPMI-1640 medium.

The siRNA targeting position 1344–1362 (CUCUCUCAUACGCGUGUAU) of human SOX18 mRNA was synthesized. A non-specific scramble siRNA sequence was used as the negative control (NC). The siRNAs were transiently transfected into MHCC-97H and HepG2 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Assays were performed 48 h after transfection.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized by using M-MuLV Reverse Transcriptase (Thermo Fisher Scientific, Rockford, IL, USA). Real-time PCR was performed using a standard SYBR-Green PCR kit (Thermo) protocol on an ABI 7300 real-time PCR machine (Applied Biosystems, Foster City, CA, USA). GAPDH served as an internal control. The gene expression was calculated using the ∆∆Ct method. All data represent the average of three replicates. The primers used are listed in Table I.

Treated and untreated MHCC-97H and HepG2 cells were lysed in ice-cold radio immunoprecipitation assay buffer. Equal amounts of protein were separated via SDS-PAGE gel electrophoresis and electro-blotted onto a nitrocellulose membrane. Immunodetection of proteins was performed using specific antibodies. Densitometric analysis was performed with Image J software. Primary antibodies were obtained from the following companies: i) SOX18, PDGFB, IGF1R and CCL3 (Abcam, Cambridge, MA, USA); ii) CCL2, CCL5, RhoA and GAPDH, CST (Biotech, Danvers, MA, USA).

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) (Dojindo Lab, Kumamoto, Japan). Briefly, the treated and untreated MHCC-97H and HepG2 cells were seeded onto 96-well plates. At the indicated time-point, CCK8 solution was added to each well and incubated for 1 h. The optical density values (OD) were measured at 450 nm by using a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). All experiments were performed in triplicates and repeated at least three times.

Propidium iodide (PI) staining was used to analyze DNA content. Treated and untreated MHCC-97H and HepG2 cells were harvested and labeled with PI by using previously described methods (20). DNA content was analyzed using a FACScan flow cytometry (BD Biosciences, San Jose, CA, USA) and the percentages of cells in the G0/G1, S and G2/M phases were determined with the FlowJo software (Tree Star). Experiments were performed in triplicate.

The percentage of apototic cells was determined by double staining with Annexin V-fluorescein isothiocyanate (FITC) and PI. With or without siRNA transfection for 48 h, both adherent and floating cells were harvested, washed with PBS, pelleted and re-suspended in an incubation buffer containing Annexin V-FITC (BD Biosciences) and PI. The subsequent analysis was performed on a FACScan flow cytometry.

Quantitative cell migration and invasion assays were performed using 12-well Boyden chambers (Coring, NY, USA). For the migration assay, siRNA-transfected cells were serum-starved for 24 h, and then 5×10⁴ cells were seeded into the upper well of the Boyden chambers, with 500 ml of serum-free medium added to the lower chamber. After 24 h of incubation, the cells on the upper surface of the filter were completely removed. The remaining cells were washed with PBS, fixed in 4% paraformaldehyde and stained with 0.2% crystal violet. The migrated cells were observed under a Leica inverted microscope (Deerfield, IL, USA) and counted.

For the in vitro invasion assay, the upper well of the Boyden Chamber was pre-coated with 10 mg/ml Matrigel (BD Biosciences). The rest of the assay was performed as described above.

To gain further insight into the biological pathways involved in HCC pathogenesis through the SOX18 pathway, GESA, a method of analyzing and interpreting microarray and the data using biological knowledge (21), was performed using GSEA version 2.0 from the Broad Institute at MIT, as previously described (22,23). RNA-sequencing data of the HCC cohort were downloaded from The Cancer Genomic Atlas project (TCGA) and analyzed by GSEA. In the present study, GSEA firstly generated an ordered list of all genes according to their correlation with SOX18 expression and then a predefined gene set (signature of gene expression upon perturbation of certain cancer-related gene) receives an enrichment score (ES), which is a measure of statistical evidence rejecting the null hypothesis that its members are randomly distributed in the ordered list. The expression level of SOX18 was used as phenotype label and 'Metric for ranking genes' was set to Pearson's correlation. The KEGG gene set biological process database (c2.KEGG. v4.0) from the Molecular Signatures Database was used for enrichment analysis.

Survival curves were obtained by the Kaplan-Meier method and the differences in survival between low and high SOX18 expression groups were analyzed with the log-rank test. The two-tailed Student's t-test was used to evaluate statistical differences between two groups. Statistical significance was set at P<0.05. Where appropriate, data are expressed as mean ± SD.

We first measured the SOX18 mRNA level in 75 patient HCC and adjacent non-tumorous tissues by real-time PCR. As shown in Fig. 1A, statistical analysis using the Student's t-test showed that SOX18 mRNA was significantly overexpressed in the HCC tissues when compared with that in the normal tissues (P<0.001).

In addition, we re-analyzed high throughput RNA-sequencing data of the HCC cohort of TCGA and also found a significant increase in SOX18 expression in the HCC tissues, and high SOX18 expression was compared with that in the normal tissues (Fig. 1B).

We next carried out Kaplan-Meier survival analysis to investigate the clinical outcome of HCC patients with low or high SOX18 expression. As shown in Fig. 1C, the survival time of the patients with high-SOX18-expressing tu mors was significantly shorter than that of patients with low-SOX18-expressing tumors (P<0.01). These results indicated that SOX18 expression was upregulated in the HCC tissues and was correlated with poor survival rate of these patients.

To investigate the functions of SOX18 overexpression on HCC, we knocked down its expression in HCC cells by RNAi. We determined the protein and mRNA levels of SOX18 in five HCC cell lines, BEL-7404, MHCC-97H, MHCC-97L, HepG2 and SMC-7721, by western blot analysis and real-time PCR, respectively. Higher protein and mRNA levels of SOX18 were observed in two cell lines, MHCC-97H and HepG2 (Fig. 1D and E), which were selected for the RNAi experiment.

One siRNA targeting human SOX18 (SOX18-siRNA) and a negative control (NC, a non-specific scramble siRNA) were synthesized and used to transfect the MHCC-97H and HepG2 cells. The silencing effect of the siRNA was then evaluated by western blot analysis and real-time PCR. Our results indicated that SOX18-siRNA was able to efficiently suppress endogenous SOX18 expression in the MHCC-97H (Fig. 1F and G) and HepG2 cells (Fig. 1H and I).

To examine the effects of SOX18 silencing on the proliferation of HCC cells, the CCK-8 assay was performed. Cell proliferation of MHCC-97H (Fig. 2A) and HepG2 cells (Fig. 2B) transfected with SOX18-siRNA was notably impaired when compared to cell proliferation in the corresponding WT and NC cells. These results indicate that SOX18 may promote the proliferation of HCC cells.

The possible inhibitory effect of SOX18 knockdown on cell cycle progression was then evaluated. PI staining and flow cytometric analysis revealed that silencing of SOX18 in the MHCC-97H (Fig. 2C) and HepG2 cells (Fig. 2D) caused an increase in cells in the G1 phase and a corresponding decrease in cells in the S and G2/M phases. We also assessed the apoptotic function of SOX18 in the HCC cells by Annexin V-FITC/PI staining assay. As shown in Fig. 2E and F, MHCC-97H and HepG2 cells transfected with SOX18-siRNA exhibited slightly induced cell apoptosis compared with WT or NC cells. These results imply that the proliferation-promoting effect of SOX18 is mainly mediated by promoting cell cycle progression.

To explore the involvement of SOX18 in cell motility, Transwell assays were carried out to quantitatively determine the effect of SOX18 on cell migration. As shown in Fig. 3A, similar numbers of WT and NC cells migrated to the lower face of the Transwell membrane (MHCC-97H: WT, 304±6; NC, 301±8; HepG2: WT, 323±8; NC, 322±6), whereas the SOX18-knockdown cells exhibited a strongly inhibited motility, with <40% cells migrating (MHCC-97H: 113±6; HepG2: 125±5).

We also investigated whether SOX18 affects the invasive ability of HCC cells by an in vitro invasion assay. As shown in Fig. 3B, depletion of SOX18 dramatically reduced the cell invasive ability when compared with that of the WT and NC cells. The number of invaded knockdown cells was ~43% of that of the control cells (MHCC-97H: WT, 108±9; NC, 102±10; SOX-siRNA, 44±5; HepG2: WT, 93±6; NC, 95±7; SOX-siRNA, 41±4). These data suggest that SOX18 promotes HCC cell invasion.

To probe the SOX18-associated pathways on an unbiased basis, we performed GSEA using high throughput RNA-sequencing data of the HCC cohort from The Cancer Genome Atlas project (TCGA). GSEA is designed to detect coordinated differences in expression of predefined sets of functionally related genes. Among all the 188 predefined 'KEGG pathway' gene sets, the focal adhesion pathway and chemokine signaling pathways were identified as having a significant association with SOX18 expression in the HCC dataset (Fig. 4A and B).

The effects of SOX18 siRNA on the mRNA and protein levels of focal adhesion pathway genes (RhoA, PDGFB and IGF1R) and chemokine signaling pathway genes (CCL2, CCL3 and CCL5) were investigated. As shown in Fig. 4C–H, SOX18 siRNA treatment in the MHCC-97H and HepG2 cells significantly decreased the mRNA and protein levels of the detected genes in comparison with levels in the WT and NC groups.