Fig 2: HPSE1 overexpression discriminates OSCC and normal samples and predicts low patient survival. (A) Kaplan–Meier cumulative curves for disease-free survival of patients with OSCC as a function of HPSE1 expression, showing a 70% probability of death in patients with higher expression of HPSE1 compared with those with low expression. (B) Receiver operating characteristic (ROC) curves showing the ability of the HPSE1 overexpression distinguishes OSCC tumors from normal oral samples. (C) Kaplan–Meier of multivariate survival analysis combining HPSE1 expression levels with pT stages, where score 1: HPSE1 lower expressing samples associated with pT1/2, score 2: HPSE1 lower expressing samples associated with pT3/4 or higher HPSE1 with pT1/2, and score 3: HPSE1 higher expressing samples associated with pT3/4. These results confirmed that HPSE1 overexpression was significantly associated with higher grade tumors.

Fig 3: Variations in (A) the glycocalyx of endothelial cells (white arrows) and (B and C) HPA protein levels in infarct areas in the three groups. *P<0.05 vs. SHAM. SHAM, pigs treated with saline solution; NCR, nicorandil; ALP, alprostadil; HPA, heparinase.

Fig 4: Overexpression of HPSE1 is associated with migration, invasion, ECM remodeling, and acquisition of EMT properties. (A) Photomicrographs of cell lines were taken 0, 24, and 48 h after wounding (40X). The average width of the lacunae was measured. Migration of SCC-9 cells was significantly decreased in HPSE1-silenced cells (ShRNA HPSE1-) and increased in HPSE1-upregulated clones (OrfRNA HPSE1+). Migration analysis based on this assay showed that cells with lower expression of HPSE1 closed the scratch wound significantly slower than the SCC9 Control cells. (B) Invasion of SCC-9 cells was significantly inhibited by HPSE1 knockdown, and significantly enhanced after its upregulation. (C) Analysing EMT markers, the downregulation of HPSE1 significantly induced the expression of E-Cadherin (E-CAD), while OrfRNA HPSE1+ SCC9 clones overexpressing heparanase had a significant increasing of Vimentin (VIM) and SNAIL expressions. (D) The upregulation of HPSE1 significantly enhanced the expressions of MMP2 and MMP9. All the graphs compile experimental triplicate analyses, and the results were obtained by ANOVA followed by Tukey assay, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 5: HPSE1 is overexpressed in OSCCs patient samples and OSCC-derived cell lines. Total RNA from fresh samples and cell lines was converted to cDNA and subjected to qPCR. For gene expression analysis of tissue samples relative quantification was based on the comparison of a pool of five normal oral tissues, while the spontaneously immortalized but non-transformed epithelial cell line HGK was used as reference for comparison with OSCC-derived cell lines. The levels of HPSE1 mRNA were significantly higher in OSCC cell lines compared with HGK cells (A). The high expression levels of protein HPSE1 were confirmed on OSCC-derived cell lines by Western Blot analysis (B). The levels of HPSE1 mRNA were also significantly higher in OSCC tissue samples compared to normal oral mucosa (C). Representative images in a high-power field (200×) of immunohistochemical analysis for HPSE1 in Normal oral tissue (D) and OSCC tissue preparations confirmed its higher expression at protein level; with a distinct cytoplasmic distribution and intensity in oral cancer samples expressing both higher (E) and lower (F) levels of HPSE1. Results were statistically determined by ANOVA followed by Tukey multiple comparison test, where **p < 0.005, ***p < 0.001, and ****p < 0.0001.