Fig 1: F. nucleatum promotes LSCC tumor proliferation and metastasis by suppressing TGFBR2 expression(A) Representative data of tumors derived from AMC-HN-8 cells subjected to different experimental conditions (n = 5 for each mouse group).(B) Statistical analysis of tumor volumes from mice subjected to different experimental conditions (n = 5 for each group) as calculated with the ordinary two-way ANOVA.(C) Statistical analysis of tumor weights from mice subjected to different experimental conditions (n = 5 for each group) as calculated with the Mann-Whitney test.(D) Representative immunohistochemical staining (10 × magnification) for ADH1B and TGFBR2 expression in tumors from mice subjected to different experimental conditions. Scale bar, 100 μm.(E) Representative data of hematoxylin-eosin (H&E)-stained sections of metastatic nodules from lungs.(F) Statistical analysis of the number of metastatic nodules in lungs from mice subjected to different experimental conditions (n = 5 for each group) as calculated with the Mann-Whitney test.(G) Representative data of metastatic nodules in the neck (n = 2, left) and H&E-stained sections (40 × magnification, right) from mice in the F. nucleatum group. Scale bar, 500 μm.(H) Representative data of metastatic nodules in the right thigh (n = 1, left) and H&E-stained sections (40 × magnification, right) from mice in the F. nucleatum group. Scale bar, 500 μm.(I) Representative data of metastatic nodules in the right thigh (n = 1, left) and H&E-stained sections (40 × magnification, right) from mice in the F. nucleatum + TGFBR2 group. Scale bar, 500 μm.(J) Relative expression of miR-155-5p (left) and miR-205-5p (right) in tumors from mice subjected to different experimental conditions (n = 5 for each group) as calculated with the Mann-Whitney test.(K) Representative fluorescence in situ hybridization (FISH) assays are shown (400 × magnification) for miR-155-5p (Cy3-labeled, left) and miR-205-5p (Cy3-labeled, right) expression in LSCC cells cultured in the presence or absence of F. nucleatum (FUS664, FAM-labeled). Scale bar, 2 μm. Data are represented as mean ± SD.
Fig 2: The F. nucleatum content correlates with the miR-155-5p, miR-205-5p, ADH1B, and TGFBR2 levels in LSCC and indicates poor oncologic outcome(A–C) Statistical analysis of the F. nucleatum amount in samples from patients in Cohort 3 as calculated by the Mann-Whitney test.(D) Receiver operating characteristic (ROC) analysis was conducted based on the amount of F. nucleatum and the eighth AJCC criteria for LSCC in samples from patients in Cohort 3.(E) DFS was determined in patients with LSCC from Cohort 3, who were stratified by high or low F. nucleatum content based on the cutoff value as assessed by the log rank test.(F) CSS was determined in patients with LSCC from Cohort 3, who were stratified by high or low F. nucleatum content based on the cutoff value as assessed by the log rank test.(G) Univariate analysis (top) and multivariate analysis (down) for DFS were performed on patients from Cohort 3. The bars correspond to 95% confidence intervals.(H) The correlation between F. nucleatum content and the combined expression level of miR-155-5p and miR-205-5p as assessed by the two-tailed nonparametric Spearman correlation coefficient.(I) The correlation between F. nucleatum content and ADH1B (left) and TGFFBR2 (right) expression in patients from Cohort 3 as assessed by the two-tailed nonparametric Spearman correlation coefficient.(J) Representative FISH assays (400 × magnification) to identify common bacteria-specific 16S rRNA sequences (EUB338, Cy3-labeled), F. nucleatum (FUS664, FAM-labeled), miR-155-5p (Cy3-labeled), and miR-205-5p (Cy3-labeled) in recurrent and nonrecurrent LSCC tissues. Scale bar, 2 μm.(K) Representative immunohistochemistry (10 × magnification) of ADH1B and TGFBR2 expression in the high and low F. nucleatum groups. Scale bar, 100 μm.(L) Schematic diagram of the relationship among F. nucleatum, ethanol metabolism reprogramming, and EMT in LSCC. Data are represented as mean ± SD.
Fig 3: F. nucleatum inhibits ADH1B and TGFBR2 expression via selective gain of miR-155-5p and miR-205-5p via the TLR4/MYD88 signaling pathway(A) The predicted binding sequences for miR-155-5p and miR-205-5p within the 3′ UTRs of human ADH1B and TGFBR2 mRNA. Seed sequences are highlighted.(B) Dual-luciferase activity was measured in three LSCC cell lines cocultured with F. nucleatum or treated with miR-155-5p and miR-205-5p mimics or control miRNA. Dual-luciferase reporters expressing wild-type (Wt) or mutant (Mut) 3′ UTRs from human ADH1B and TGFBR2 mRNA were used. The dual-luciferase activity was normalized based on the control miRNA transfection as assessed by the ordinary one-way ANOVA.(C) Western blotting was performed for ADH1B and TGFBR2 in three LSCC cell lines transfected with miR-155-5p and miR-205-5p mimics or inhibitor and cultured in the presence or absence of F. nucleatum.(D) Western blotting for ADH1B and TGFBR2 expression was performed in three LSCC cell lines transfected with TLR4 and MYD88 siRNAs and cultured in the presence or absence of F. nucleatum.(E) Statistical analysis of the relative expression of miR-155-5p (left) and miR-205-5p (right) in samples from Cohort 1 as calculated with the Wilcoxon matched pairs signed-rank test and Mann-Whitney test.(F) Correlation between the amount of F. nucleatum and the relative expression of miR-155-5p and miR-205-5p in LSCC tissues from Cohort 1 as assessed by the two-tailed nonparametric Spearman correlation coefficient. Data are represented as mean ± SD.
Fig 4: The positive feed-forward loop between F. nucleatum and ethanol metabolism reprogramming promotes LSCC aggressiveness(A) A CCK-8 assay was performed in three LSCC cell lines cultured in the presence or absence of F. nucleatum and 0.1% (v/v) ethanol as assessed by the ordinary two-way ANOVA.(B) Transwell migration assays were performed in three LSCC cell lines cultured in the presence or absence of F. nucleatum and 0.1% (v/v) ethanol as calculated with the Student’s unpaired t test.(C) Transwell invasion assays were performed in three LSCC cell lines cultured in the presence or absence of F. nucleatum and 0.1% (v/v) ethanol as calculated with the Student’s unpaired t test.(D) Statistical analysis of the amount of F. nucleatum in three LSCC cell lines cocultured with F. nucleatum and treated with 0.1% (v/v) ethanol as calculated with Student’s unpaired t test.(E) Western blotting was performed to assess ADH1B expression in three LSCC cell lines cocultured with F. nucleatum or E. coli.(F) The ethanol content was measured by colorimetric assay in three LSCC cells with or without ADH1B-overexpressing lentiviral vector transduction and subsequently cocultured with F. nucleatum and 0.1% (v/v) ethanol as assessed by the ordinary one-way ANOVA.(G) The aldehyde content was measured by colorimetric assay in three LSCC cell lines with or without transduced ADH1B-overexpressing lentiviral vector and subsequent coculture with F. nucleatum and 0.1% (v/v) ethanol as assessed by the ordinary one-way ANOVA.(H) Statistical analysis of ADH1B expression in samples from Cohort 1 as calculated with the Wilcoxon matched pairs signed-rank test and Mann-Whitney test.(I) Western blotting for ADH1B expression was performed on samples from Cohort 1.(J) The correlation between the amount of F. nucleatum and ADH1B expression in LSCC tissues of Cohort 1 as assessed by the two-tailed nonparametric Spearman correlation coefficient. Data are represented as mean ± SD.
Fig 5: Quantitative real-time PCR analysis of ethanol metabolism–related and oxidative stress genes in the liver tissue of acute ethanol-induced mice. The mRNA levels of ADH1 (a), CYP2E1 (b), ALDH2 (c), SOD2 (d), Gpx1 (e), Cox2 (f), TNF-α (g), and MCP1 (h) in the liver are shown at 3 h after ethanol injection. Expression level was normalized to that of glyceraldehyde 3-phosphate dehydrogenase. Abbreviations: ADH1, alcohol dehydrogenase 1; cytochrome p450 enzyme 2E1, CYP2E1; ALDH2, aldehyde dehydrogenase 2; SOD2, superoxide dismutase 2; Gpx1, glutathione peroxidase 1; Cox2, cyclooxygenase 2; TNF-α, Tumor necrosis factor-α; MCP1, Monocyte chemoattractant protein 1. Values represent the means ± SEM (n = 5 mice for each group). * p < 0.05 versus control, # p < 0.05 versus Veh.
Supplier Page from Abcam for Alcohol Dehydrogenase Assay Kit