Journal: Journal of Dental Sciences

Article Title: Rho GTPase activating protein 11A promotes tongue squamous cell carcinoma proliferation and is a transcriptional target of forkhead box M1

doi: 10.1016/j.jds.2024.02.015

Figure Lengend Snippet: ARHGAP11A upregulation was associated with poor prognosis in tongue squamous cell carcinoma. A. Comparison of ARHGAP11A between primary oral squamous cell carcinoma (OSCC) tissues and adjacent normal tissues in The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma (TCGA-HNSC). B. Comparison of ARHGAP11A expression in primary tumors from different anatomic sites in TCGA-HNSC. C. Comparison of ARHGAP11A between primary tongue squamous cell carcinoma (TSCC) tissues and adjacent normal tissues in TCGA-HNSC. D-E. Patients with primary OSCC (D) or tongue squamous cell carcinoma (TSCC) (E) were separated into two groups by median ARHGAP11A expression. Kaplan–Meier (K–M) survival curves were generated to compare the difference in overall survival (OS) and progression-free survival (PFI) between the higher and lower ARHGAP11A expression group. F-G. Quantitative results (F) and representative images (G) of immunohistochemistry (IHC) staining of ARHGAP11A in TSCC tumor tissues and adjacent normal tissues of a commercial TSCC tissue microarray. H. ARHGAP11A mRNA expression in multiple TSCC cell lines, using from data from Cancer Cell Line Encyclopedia (CCLE).

Article Snippet: The OSCC cell lines SCC4 and SCC25 were acquired from Procell (Wuhan, China) and cultured under the recommended medium and conditions.

Techniques: Comparison, Expressing, Generated, Immunohistochemistry, Microarray

Journal: Journal of Dental Sciences

Article Title: Rho GTPase activating protein 11A promotes tongue squamous cell carcinoma proliferation and is a transcriptional target of forkhead box M1

doi: 10.1016/j.jds.2024.02.015

Figure Lengend Snippet: Knocking down of ARHGAP11A expression impairs the proliferation of tongue squamous cell carcinoma. A-B. RT-qPCR and western blotting analyses were performed to check the expression of ARHGAP11A at the mRNA (A) and protein (B) levels in SCC4 and SCC25 cells 48 h after lentivirus-mediated ARHGAP11A knockdown. C-E. CCK-8 (C), colony formation (D) and transwell assay of migration and invasion (E) were performed to explore the influence of ARHGAP11A knockdown on cell proliferation, migration and invasion in SCC4 and SCC25 cells. F-G. SCC4 and SCC25 cells with or without lentivirus-mediated ARHGAP11A knockdown were used for in vivo studies. Xenograft tumors were removed and pictured (E) and the weights of tumors in each group (F) were calculated at the end of animal study. H. Representative immunohistochemistry (IHC) staining images of Ki-67 expression SCC4- and SCC25-derived xenograft tumors. Scale bar: 50 μm ### and ∗∗∗, P < 0.001.

Article Snippet: The OSCC cell lines SCC4 and SCC25 were acquired from Procell (Wuhan, China) and cultured under the recommended medium and conditions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Transwell Assay, Migration, In Vivo, Immunohistochemistry, Derivative Assay

Journal: Journal of Dental Sciences

Article Title: Rho GTPase activating protein 11A promotes tongue squamous cell carcinoma proliferation and is a transcriptional target of forkhead box M1

doi: 10.1016/j.jds.2024.02.015

Figure Lengend Snippet: ARHGAP11A knockdown induces G1 phase arrest by regulating multiple cell-cycle proteins in tongue squamous cell carcinoma. A-B. Representative fluorescent ubiquitination-based cell cycle indicator (FUCCI) images and ARHGAP11A staining (A) and quantitation of ARHGAP11A mRNA and protein expression in different phases with the cell-cycle (B) in U2OS cells in the human protein atlas (HPA). Image credit: the HPA, data were retrieved from: https://www.proteinatlas.org/ENSG00000198826-ARHGAP11A/subcellular#cell_cycle . C. Western blotting analysis was performed to check the expression of indicated proteins in SCC4 and SCC25 cells 48 h after lentivirus-mediated ARHGAP11A knockdown. D-E. Representative images (D) and quantitation (E) of cell-cycle distribution of SCC4 and SCC25 cells with or without lentivirus-mediated ARHGAP11A knockdown. ∗∗∗, P < 0.001.

Article Snippet: The OSCC cell lines SCC4 and SCC25 were acquired from Procell (Wuhan, China) and cultured under the recommended medium and conditions.

Techniques: Knockdown, Ubiquitin Proteomics, Staining, Quantitation Assay, Expressing, Western Blot

Journal: Journal of Dental Sciences

Article Title: Rho GTPase activating protein 11A promotes tongue squamous cell carcinoma proliferation and is a transcriptional target of forkhead box M1

doi: 10.1016/j.jds.2024.02.015

Figure Lengend Snippet: ARHGAP11A is transcriptionally activated by FOXM1 in tongue squamous cell carcinoma cells. A. A heatmap showing the general expression profile of ARHGAP11A and the candidate transcription factors and chromatin regulators with potential binding to the ARHGAP11A promoter region (within 10 kb before the transcription start site) in the primary tongue squamous cell carcinoma (TSCC) cases in TCGA-HNSC. B. A dot chart showing the correlation between ARHGAP11A and FOXM1 in the primary TSCC cases. C-E. RT-qPCR (C–D) and western blotting (E) analyses were performed to check the expression of FOXM1 and ARHGAP11A at the mRNA and protein levels in SCC4 and SCC25 cells 48 h after lentivirus-mediated FOXM1 knockdown. F. Dual-luciferase assays were performed to check the relative luciferase activities of pGL3-11A-pWT-1 in SCC4 and SCC25 cells with or without FOXM1 knockdown. G. A schematic diagram showing the design of recombinant pGL3-basic plasmids carrying integrated ARHGAP11A promoter region (pGL3-11A-pWT-1) and the truncated fragments. H–I. Dual-luciferase assays were performed to check the relative luciferase activities of pGL3-11A-pWT-1 and the truncated counterparts in SCC4 and SCC25 cells. n.s., not significant; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: The OSCC cell lines SCC4 and SCC25 were acquired from Procell (Wuhan, China) and cultured under the recommended medium and conditions.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Western Blot, Knockdown, Luciferase, Recombinant

Journal: Cancer Gene Therapy

Article Title: Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial–mesenchymal transition-induced angiogenesis

doi: 10.1038/cgt.2016.30

Figure Lengend Snippet: Angiopoietin 2 (ANG2) was upregulated in oral squamous cell carcinoma (OSCC) tissues. ( a ) Immunohistochemical and quantification analysis of ANG2 protein expression in normal liver tissue (up) and OSCC (bottom) samples using specific anti-ANG2 antibody. Representative photographs were obtained at × 200 magnification. ( b ) ANG2 mRNA expression levels in OSCC tissue samples and normal tissue samples (12 cases) were examined by real-time quantitative reverse transcriptase (qRT-PCR), respectively. ( c ) The expression of ANG2 in OSSC cell lines (TCA8113, cal27, SCC4, SCC15 and SCC25). GAPDH was used as an internal quantitative control. Three independent experiments were performed, and the data represent the means±s.d. * P <0.05.

Article Snippet: The human OSCC cell lines TCA8113, cal27, SCC4, SCC15 and SCC25 were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Immunohistochemical staining, Expressing, Reverse Transcription, Quantitative RT-PCR, Control

Journal: Cancer Gene Therapy

Article Title: Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial–mesenchymal transition-induced angiogenesis

doi: 10.1038/cgt.2016.30

Figure Lengend Snippet: Effects of angiopoietin 2 (ANG2) on apoptosis and the cell cycle in oral squamous cell carcinoma (OSCC) cells. ( a ) Proliferation rates were determined by the MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay on days 1–3 in TCA8113 mock cells or cells transfected with ANG2-encoded plasmids, ANG-targeted short interfering RNA (siRNA) or nonspecific (NC) siRNA. Before performing the MTS assays, cells were transfected with the indicated plasmids or siRNA for 24 h. Three independent experiments were performed, and the data represent the means±s.d. Treated and control groups of TCA8113 cells were used in subsequent experiments. ( b ) Representative dot plots showing fluorescence channel analysis of the treated and control groups of TCA8113 cells after dual staining with Annexin V and propidium iodide (PI) and analysis by flow cytometry. Columns represent the mean of three individual experiments; Bars, s.d. ( c ) Cell cycle distributions of the treated and control groups of TCA8113 cells were analyzed by flow cytometry. Data represent the mean±s.d. of three independent experiments.

Article Snippet: The human OSCC cell lines TCA8113, cal27, SCC4, SCC15 and SCC25 were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Small Interfering RNA, Control, Fluorescence, Staining, Flow Cytometry

Journal: Cancer Gene Therapy

Article Title: Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial–mesenchymal transition-induced angiogenesis

doi: 10.1038/cgt.2016.30

Figure Lengend Snippet: Overexpression of angiopoietin 2 (ANG2) increased the migration and invasion of TCA8113 cells by regulating epithelial–mesenchymal transition (EMT). ( a ) Cell migration and invasion were observed using a transwell chamber. ( b ) Western blot analysis of the cell proliferation-related proteins vimentin, Snail, Twist and E-cadherin in treated and control groups of TCA8113 cells. GAPDH served as a loading control. ( c ) Enzyme-linked immunosorbent assay (ELISA) was performed to analyze VEGF concentrations in cell-free supernatants. *Compared to Crtl or NC group. VEGF, vascular endothelial growth factor.

Article Snippet: The human OSCC cell lines TCA8113, cal27, SCC4, SCC15 and SCC25 were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Over Expression, Migration, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer Gene Therapy

Article Title: Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial–mesenchymal transition-induced angiogenesis

doi: 10.1038/cgt.2016.30

Figure Lengend Snippet: Overexpression of angiopoietin 2 (ANG2) promoted tumorigenicity, increased angiogenesis and reduced apoptosis in nude mice. Subcutaneous injection of treated and control groups of TCA8113 cells into nude mice. ( a , b ) Tumor volume was examined every week. Three independent experiments were performed, and the data represent the means±s.d. * P <0.05. ( c ) Tumor tissues from nude mice 4 weeks after injection were subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Green TUNEL-positive cells are apoptotic cells with fragmented DNA, whereas blue Hoechst-positive cells represent all cells in this assay. Magnification is × 200. ( d ) Immunohistochemical and quantification analysis of CD31 protein expression in the treated and control groups of oral squamous cell carcinoma (OSCC) tissues with specific anti-CD31 antibody. Representative photographs were obtained at × 200 magnification. ( e ) Western blot analysis of tumor formation-related proteins including Bim, PUMA, Bcl-2, Bax, Cyclin D1 and PCNA in the treated and control groups of OSCC tissue samples. GAPDH served as a loading control.

Article Snippet: The human OSCC cell lines TCA8113, cal27, SCC4, SCC15 and SCC25 were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Over Expression, Injection, Control, TUNEL Assay, Immunohistochemical staining, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: Role of the EZH2/miR-200 axis in STAT3-mediated OSCC invasion

doi: 10.3892/ijo.2018.4293

Figure Lengend Snippet: STAT3 contributes to invasion and migration of OSCC cells. (A) Western blot detection of STAT3, p-STAT3 (Tyr705), MMP2 and MMP9 expression. GAPDH was used as a loading control. (B) MTT analysis was used to determine the IC 50 values for both OSCC cell lines. (C) Targeting STAT3 significantly inhibited SCC25 and SCC15 cell invasion and migration ability, as determined by Transwell assay (magnification, ×40). (D) Cell migration ability was measured by a wound-healing assay (magnification, ×40). *** P<0.001 vs. EV group; ### P<0.001 EV + Stattic group vs. STAT3 group. EV, empty vector; IC 50 , half maximal inhibitory concentration; MMP, matrix metalloproteinase; OSCC, oral squamous cell carcinoma; p-STAT3, phosphorylated-STAT3; STAT3, signal transducer and activator of transcription 3.

Article Snippet: SCC25 and SCC15 cell lines were transfected with NC siRNA or siRNA against EZH2 (20 nM; Guangzhou Ribobio Co., Ltd., Guangzhou, China), which were labeled as si-NC (5′-UUCUCCGAACGUGUCACGU-3′) or si-EZH2 (#1, 5′-GCUGGAAUCAAAGGAUACA-3′; #2, 5′-GTGCCCTTGTGTGATAGCACAA-3′; #3, 5′-GGCACT TTCATTGAAGAACTAA-3′), respectively, using Lipo-fectamine 2000 reagent, according to the manufacturer's protocol.

Techniques: Migration, Western Blot, Expressing, Control, Transwell Assay, Wound Healing Assay, Plasmid Preparation, Concentration Assay

Journal: International Journal of Oncology

Article Title: Role of the EZH2/miR-200 axis in STAT3-mediated OSCC invasion

doi: 10.3892/ijo.2018.4293

Figure Lengend Snippet: EZH2/miR-200/b/a/429 axis regulates the invasiveness of OSCC cells in vitro . (A) Following transfection with EZH2 siRNAs (si#1 and si#2) for 3 days, the expression levels of EZH2 were detected using western blotting. (B) OSCC cells underwent western blot analysis using antibodies specific to STAT3, p-STAT3 (Tyr705), EZH2, H3K27me3, H3 and GAPDH. EZH2 siRNA markedly inhibited EZH2 and H3K27me3 expression. In addition, p-STAT3 (Tyr705), but not STAT3, was suppressed by EZH2 depletion. (C) EZH2 siRNA induced miR-200b/a/429 expression. (D) Results of a Transwell assay demonstrated that EZH2 knockdown markedly attenuated migration (without Matrigel) and invasion (with Matrigel) of SCC25 and SCC15 cells (scale bar, 100 μ m; magnification, ×40). (E) Scratch assay demonstrated that EZH2 silencing markedly delayed wound healing in SCC25 and SCC15 cells (magnification, ×40). *P<0.05 and *** P<0.001 vs. si-NC group. EZH2, enhancer of zeste homolog 3; H3, histone 3; H3K27me3, trimethylation of lysine 27 in H3; miR-200b/a/429, microRNA-200b, -200a and -429; NC, negative control; OSCC, oral squamous cell carcinoma; p-STAT3, phosphorylated-STAT3; siRNA/si, small interfering RNA; STAT3, signal transducer and activator of transcription 3.

Article Snippet: SCC25 and SCC15 cell lines were transfected with NC siRNA or siRNA against EZH2 (20 nM; Guangzhou Ribobio Co., Ltd., Guangzhou, China), which were labeled as si-NC (5′-UUCUCCGAACGUGUCACGU-3′) or si-EZH2 (#1, 5′-GCUGGAAUCAAAGGAUACA-3′; #2, 5′-GTGCCCTTGTGTGATAGCACAA-3′; #3, 5′-GGCACT TTCATTGAAGAACTAA-3′), respectively, using Lipo-fectamine 2000 reagent, according to the manufacturer's protocol.

Techniques: In Vitro, Transfection, Expressing, Western Blot, Transwell Assay, Knockdown, Migration, Wound Healing Assay, Negative Control, Small Interfering RNA