Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 knockdown and upregulation efficiency in OSCC cells. The efficiency of endogenous HPSE1 modulation was verified by Immunofluorescence (A) , Western Blot (B) and RT-qPCR (C) analyzes. For the loss-of-function strategy, SCC-9 cells were transduced with shRNA expressing a vector sequence against HPSE1 (shRNA HPSE1−) and empty vector (scrRNA control). For the gain-of-function strategy, SCC-9 cells were transduced with a ORF clone in a shuttle vector to enhance HPSE1 expression (orfRNA HPSE1+), along with mock-transduced cells as described in Methods . shRNA HPSE1− cells showed a significant reduction and orfRNA HPSE+ cells showed a significant upregulation of HPSE1 in both mRNA and protein levels compared to scrRNA control and parental mock-transduced SCC-9 control cells (pSCC9), without targeting sequences. Results were statistically determined by ANOVA, followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Knockdown, Immunofluorescence, Western Blot, Quantitative RT-PCR, Transduction, shRNA, Expressing, Plasmid Preparation, Sequencing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Downregulation of HPSE1 inhibits proliferation and enhances apoptosis of OSCC cells. Cells were subjected to MTT cell proliferation (A) , DNA content cell cycle analysis (B) , and apoptosis (C) assays. (A) Cell Proliferation Assay of the OSCC SCC9 cell line (Control) compared to empty vector SCC9 control clone (ScrRNA control), HPSE1 inhibitory/silencing SCC9-ShRNA HPSE1− clone, and the HPSE1 overexpressing SCC9-OrfRNA HPSE1+ clone. (B) Quantification of cell cycle analysis was performed by flow cytometry after staining with propidium iodide for the SCC-9 control cells and the respectives HPSE1-modulated clones. Abrogation of HPSE1 induced arrest of the SCC-9 cells cycle in G1 phase, and its upregulation significantly induced cell proliferation. (C) Flow cytometric analysis of apoptosis showed a remarkable increase in the number of apoptotic cells in HPSE1-silenced cells (ShRNA HPSE1−), while SCC-9 overexpressing HPSE1 (Orf-RNA HPSE1+) exhibited a reduction in apoptosis. (D) No significant differences in cell adhesion properties were observed among any transduced clones modulating HPSE1 expression, compared to parental mock-transduced cells (control). Plots compose experimental triplicate analysis and were statistically calculated using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: MTT Cell Proliferation, Cell Cycle Assay, Proliferation Assay, Control, Plasmid Preparation, shRNA, Flow Cytometry, Staining, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: 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.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Migration, shRNA, Clone Assay, Expressing, Control, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 enhances tumor neo-vascularization and induces VEGFA expression. HUVEC cells on a Miogel 2D layer co-culture. Photomicrograph of HUVEC endothelial cells added with preconditioned medium from OSCC cell lines and their respective clones after 12 h of experiment: (A) HUVEC cells incubated with preconditioned medium of SCC9-OrfRNA HPSE1+ clone overexpressing HPSE1 (100×); (B) HUVEC cells incubated with preconditioned medium of parental SCC9 (Control) (100×); (C) HUVEC cells incubated with preconditioned medium of SCC9-ShRNA HPSE1- clone with reduction of HPSE1 expression (100×). (D) Quantification of measures of the vessel circumference perimeter for each one of the conditions; the graph compiles two experimental triplicate analysis. (E) Gene expression analysis of endothelial growth factor VEGFA by qRT-PCR; the graph compiles folded expression values of relative quantification by ddCT obtained through comparison of clones OrfRNA HPSE1+ and ShRNA HPSE1− relative to the control SCC9 cells (normal reference = 1). Normalization of the analysis was performed using the endogenous control gene, PPIA. Statistical tests were performed using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing, Co-Culture Assay, Clone Assay, Incubation, Control, shRNA, Gene Expression, Quantitative RT-PCR, Quantitative Proteomics, Comparison

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: Human liver hepatocellular cells (HepG2) were stimulated in the presence or absence of 2 μM S1P for 72 h. ( A ) S1P increased mRNA expression of S1P 1 . HepG2 cells were treated with S1P 1 inhibitor W146 (10 μM), HPSE inhibitor low molecular weight heparin (50 μg/ml), generic MMP inhibitor Ilomastat (GM6601, 10 μM), or GM6001 negative control (NC) for 30 min before S1P stimulation for 72 h. ( B ) S1P downregulated mRNA transcripts of syndecan-1, which was blocked by W146 and heparin, but was not by Ilomastat. ( C ) S1P-induced sGAG release from HepG2 cells was eliminated by W146, heparin, and Ilomastat, respectively. sGAG in the culture medium was detected by using 1,9-dimethylmethylene blue (DMMB) assay. ( D ) S1P decreased syndecan-1 staining ( green ) but induced translocation of syndecan-1 into the nucleus (DAPI, blue ). Confocal images depict a z -plane through the center of the cell nucleus. Scale bar, 10 μm. ( E ) and ( F ), a representative image of Western blots (E) with densitometric quantification (F) of syndecan-1 in cell lysates after 30 min exposure of W146, heparin, and Ilomastat before S1P treatment for 72 h. Results are presented as a ratio to GAPDH. ( G ) and ( H ), immunofluorescence images (maximum-intensity projections of a confocal z -stack) (G) and the coverage of heparan sulfate (HS, green ). Nuclei were counterstained with DAPI ( blue ) (H). * P < 0.05 as compared with CT; # P < 0.05 as compared with S1P (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: Expressing, Molecular Weight, Negative Control, Dimethylmethylene Blue Assay, Staining, Translocation Assay, Western Blot, Immunofluorescence

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HPSE knockdown (HPSE shRNA) and MMP-7 knockdown (MMP-7 shRNA) by shRNA transduction were performed. Then, cells were treated with S1P 1 inhibitor W146 (10 μM), and HPSE inhibitor low molecular weight heparin (50 μg/ml) for 30 min before S1P stimulation for 72 h. Cells transfected with scrambled shRNA were set as a negative control (NC). HPSE expression and activity were modulated by S1P via S1P 1 . ( A ) HPSE mRNA expression levels in WT and HPSE shRNA cells were determined by qRT-PCR. The data were corrected to GAPDH as an internal control and represents the mean ± S.D. (Error bars) of at least three independent experiments performed in triplicate, and final results were normalized to the non-treated WT control cells. ( B ) and ( C ), HPSE activity in protein extracts (B) and conditioned media (C) in WT and HPSE shRNA cells. HPSE activity is expressed as HS removed rate, nanograms per minute. ( D ) MMPs activity in the culture medium was determined by using Sensolyte 390 generic MMP activity kit (AnaSpec). The results were normalized to the WT control cells. ( E ) MMP-7 mRNA expression levels in WT, HPSE shRNA, and MMP-7 shRNA cells. ( F ) MMP-7 activity in the culture medium was determined by using SensoLyte 520 MMP-7 assay kit (AnaSpec). MMP-7 shRNA and WT control cells were treated with 2 μM S1P for 72 h. Then, sGAG assay, syndecan-1 expression, and staining of HS were performed. ( G ) sGAG in the culture medium was measured by DMMB assay. ( H ) a representative image of Western blots with densitometric quantification of syndecan-1. ( I ), the coverage of heparan sulfate (HS) was estimated by using maximum-intensity projections of a confocal z -stack. * P < 0.05, ** P < 0.01 as compared with CT; # P < 0.05 as compared with S1P-treated WT control (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: Knockdown, shRNA, Transduction, Molecular Weight, Transfection, Negative Control, Expressing, Activity Assay, Quantitative RT-PCR, Control, Staining, Dimethylmethylene Blue Assay, Western Blot

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HepG2 WT and HPSE shRNA cells were stimulated with 2 μM S1P for 72 h with or without pretreatment for 30 min with S1P 1 inhibitor W146 (10 μM), PI3K/AKT inhibitor LY294002 (20 μM) or ERK1/2 inhibitor GDC-0994 (50 μM). ( A ) and ( B ), the phosphorylation levels of AKT (A) and ERK1/2 (B) in WT and HPSE shRNA cells were evaluated by Western blot. Histograms show densitometric quantification of p -AKT or p -ERK normalized to total AKT or ERK, respectively. ( C ) LY294002 inhibited the S1P-induced expression of HPSE mRNA, but GDC-0994 did not. ( D ) and ( E ) LY294002 abolished the S1P-induced mRNA expression and activity of MMP-7, but GDC-0994 did not. The mRNA expression of MMP-7 in cell lysates was performed by qRT-PCR, and the activity of MMP-7 in cell mediums was determined by using SensoLyte 520 MMP-7 assay kit (AnaSpec). ( F ) S1P-induced sGAG release was blocked by LY294002 or in partly inhibited by GDC-0994. sGAG in culture medium was detected by DMMB assay. ( G ) and ( H ), effects of S1P in the suppression of syndecan-1 expression (G) and decrease in the coverage of HS (H) were blocked by LY294002 or in partly inhibited by GDC-0994. Syndecan-1 expression was measured by Western blot. The coverage of heparan sulfate (HS) was estimated by using maximum-intensity projections of a confocal z -stack. * P < 0.05 as compared with WT control cells; # P < 0.05 as compared with S1P-treated WT control (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: shRNA, Phospho-proteomics, Western Blot, Expressing, Activity Assay, Quantitative RT-PCR, Dimethylmethylene Blue Assay, Control

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HepG2 WT, HPSE shRNA, and syndecan-1 shRNA cells were treated with 2 μM S1P for 72 h with or without pretreatment for 30 min with S1P 1 inhibitor W146 (10 μM), ERK1/2 inhibitor GDC-0994 (50 μM), or 5 μg/ml neutralizing TGF-β1 antibody as indicated. ( A ), ( B ), and ( C ), S1P promoted TGF-β1 secretion in culture medium (A), and mRNA (B) and protein expression (C) as measured by ELISA, qRT-PCR and Western blot assay, respectively. Representative image of Western blots with densitometric quantification are shown for TGF-β1, and the results represent the mean ± S.D. as a ratio to GAPDH. ( D ) the knockdown of syndecan-1 by shRNA transduction was checked. HepG2 cells were stimulated with 0.5 ng/ml exogenous TGF-β1 for 72 h. ( E ) change in expression of HPSE mRNA in cells treated with TGF-β1 was not detected. HepG2 cells were pretreated with ERK1/2 inhibitor GDC-0994 (50 μM) for 30 min before exposure to 0.5 ng/ml exogenous TGF-β1 for 72 h. ( F ) TGF-β1 not significantly changed syndecan-1 mRNA expression. HepG2 WT and MMP-7 shRNA cells were stimulated with 0.5 ng/ml exogenous TGF-β1 for 72 h. ( G ) knockdown of MMP-7 abolished the TGF-β1-induced sGAG release. * P < 0.05, ** P < 0.01 as compared with WT control cells; # P < 0.05 as compared with S1P-treated WT control (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Knockdown, Transduction, Control

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HepG2 control and syndecan-1 shRNA cells were treated with 2 μM S1P for 72 h with or without an addition of 5 μg/ml neutralizing TGF-β1 antibody. ( A ) and ( B ), HepG2 cells knocked down for syndecan-1 (syndecan-1 shRNA) compared with control cells were plated to invade through Matrigel-coated Transwell. An addition of 5 μg/ml neutralizing TGF-β1 antibody abolished S1P-induced cell invasion. The invaded cells were quantified and representative images are present. ( C ), ( D ), and ( E ), representative images were photographed under a scanning electron microscope (C). Alterations in cell area (D) and morphology (the aspect ratio and circularity, E) were evaluated by using image J. The results represent the mean ± S.D. of 80 cells from at least three images. ( F ) expression of epithelial phenotype marker E-cadherin and mesenchymal phenotype marker Vimentin, as well as transcription regulatory protein Snail, were analyzed by Western blotting with densitometric quantification. ( G ) a schematic summary of S1P-induced EMT via an MMP-7/syndecan-1/TGF-β1 autocrine loop. This pathway involves activation of PI3K/AKT signaling pathways via S1P 1 , which triggers an enzyme HPSE, leading to an increase in expression and activity of MMP-7 and leading to activation of the ERK1/2 signaling pathway. MMP-7 mediated the shedding of syndecan-1. ERK1/2 mediates the suppression of syndecan-1. The loss of syndecan-1 causes an increase in TGF-β1 secretion and expression. The limited protracted increase in TGF-β1 further enhances MMP-7 activity, leading to syndecan-1 shedding, thus forming an MMP-7/syndecan-1/TGF-β1 autocrine loop. Finally, TGF-β1 and syndecan-1 are essential for S1P-induced epithelial to mesenchymal transition. Syndecan-1 probably behaves as a brake of the autocrine loop and an operator modulated forming of the metastasis-permissive microenvironment. * P < 0.05 as compared with WT control cells (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: Control, shRNA, Microscopy, Expressing, Marker, Western Blot, Activation Assay, Protein-Protein interactions, Activity Assay