Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4 is significantly expressed in esophageal squamous cell carcinoma (A) Gene distribution showing m6A methylation sites identified through MeRIP-seq analysis. (B) Distribution pattern of m6A enrichment peaks. (C) Volcano plot analysis of differentially expressed membrane proteins from transcriptome sequencing data (|log2 FC| ≥ 2 and P ≤ 0.05). (D) Venn diagram depicting the overlap between differentially expressed membrane protein genes from transcriptome sequencing and genes with CDS region methylation identified by MeRIP-seq. (E) RT-qPCR validation comparing expression levels of Nectin-4, GJB3, and DSG2 between normal esophageal epithelium and esophageal squamous carcinoma cell lines. (F) Nectin-4 expression analysis in esophageal cancer using the GEPIA2 online database. (G) Western blot analysis comparing Nectin-4 protein levels between five ESCC cell lines (TE-1, KYSE-30, KYSE-150, KYSE-410, and KYSE-510) and normal esophageal epithelial cells (HET-1A). *P < 0.05; **P < 0.01.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Methylation, Membrane, Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4 facilitates the progression of esophageal squamous carcinoma (A,B) Cell proliferation effects of Nectin-4 in KYSE-410 and KYSE-510 esophageal squamous carcinoma cell lines were validated by CCK8 and colony formation assays. (C,D) Impact of Nectin-4 on migration and invasion capabilities of KYSE-410 and KYSE-510 cells was assessed through wound healing and Transwell assays. (E) In vivo effects of Nectin-4 were evaluated using a subcutaneous xenograft model in nude mice with KYSE-410 cells (n = 8). (F,G) Immunohistochemical and H&E staining for Nectin-4, Ki-67, and vimentin were performed to evaluate the effects of control and Nectin-4 knockdown on tumor growth, proliferation, and metastatic potential (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Migration, In Vivo, Immunohistochemical staining, Staining, Control, Knockdown

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: METTL3 affects the progression of esophageal squamous cell carcinoma by facilitating the stabilization of Nectin-4 via m 6 A modification (A) Designated binding sites for Nectin-4 that exhibit m6A methylation according to MeRIP-seq data. (B) The m6A methylation target database M6A2Target was used to predict online that Nectin-4 undergoes m6A methylation via associated methyltransferases. (C) The online database TIMER 2.0 was used to analyze the expression of METTL3 in esophageal cancer. (D,E) RT-qPCR and western blot analyses confirmed the expression of METTL3 in five ESCC cell lines relative to that in normal esophageal epithelial cells. (F) The SRAMP online database was used to predict the m6A methylation sites of Nectin-4. (G) Dual-luciferase activity assay validation confirmed that METTL3 facilitates the m6A methylation alteration of Nectin-4. (H) MeRIP-qPCR confirmed that METTL3 facilitates alterations in the m6A methylation of Nectin-4. (I) Actinomycin D confirmed that METTL3 influences the stability of Nectin-4 through m6A modification. (J) RT-qPCR validation of the impact of METTL3 on Nectin-4 mRNA levels. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Modification, Binding Assay, Methylation, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Biomarker Discovery

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: METTL3 facilitates the progression of esophageal squamous cell carcinoma (A,B) Cell proliferation effects of METTL3 in KYSE-410 and KYSE-510 esophageal squamous carcinoma cell lines were evaluated by CCK8 and colony formation assays. (C,D) Impact of METTL3 on migration and invasion capabilities of KYSE-410 and KYSE-510 cells was assessed through wound healing and Transwell assays. (E) In vivo effects of METTL3 were evaluated using a subcutaneous xenograft model in nude mice with KYSE-410 cells (n = 8). (F, G) Immunohistochemical staining for METTL3, vimentin, Ki-67, and Nectin-4 was performed to evaluate the effects of control and METTL3 knockdown on tumor growth, proliferation, and metastatic potential, with H&E staining used to assess METTL3-mediated metastasis (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Migration, In Vivo, Immunohistochemical staining, Staining, Control, Knockdown

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4 overexpression mitigates the suppressive effect of METTL3 knockdown on the progression of esophageal squamous cell carcinoma (A, B) CCK8 and colony formation assays demonstrated that Nectin-4 overexpression attenuated the anti-proliferative effects of METTL3 knockdown in KYSE-410 and KYSE-510 esophageal squamous cell carcinoma cell lines. (C,D) Wound healing and Transwell assays revealed that Nectin-4 expression counteracted the inhibitory effects of METTL3 knockdown on migration and invasion in KYSE-410 and KYSE-510 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Over Expression, Knockdown, Expressing, Migration

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: VNN1 facilitates the progression of esophageal squamous carcinoma (A) Heatmap showing differential gene expression following Nectin-4 knockdown transcriptome sequencing. (B) Venn diagram illustrating overlap between downregulated genes in Nectin-4-knockdown cells (log2 FC ≤ –1.5 and P ≤ 0.01) and highly expressed genes in esophageal cancers from TCGA database. (C) RT-qPCR validation comparing expression levels of VNN1, CCL28, and PLEKHS1 between normal esophageal epithelial cells and esophageal squamous carcinoma cell lines. (D) RT-qPCR analysis of VNN1,CCL28, and PLEKHS1 expression changes following Nectin-4 knockdown. (E) VNN1 expression analysis in esophageal carcinoma using GEPIA2 database. (F) Western blot analysis of VNN1 protein levels following Nectin-4 knockdown. (G) Western blot analysis comparison of VNN1 protein expressions between normal esophageal epithelium and esophageal squamous carcinoma cell lines. (H) KEGG pathway enrichment analysis of downregulated genes from transcriptome sequencing. (I,J) Cell proliferation effects of METTL3 in KYSE-410 and KYSE-510 cells assessed by CCK8 and colony formation assays. (K,L) Impact of METTL3 on cell migration and invasion evaluated through wound healing and Transwell assays. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Gene Expression, Knockdown, Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot, Comparison, Migration

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4, METTL3, and VNN1 in tissues from individuals with esophageal squamous carcinoma hold clinical importanceare clinically important (A) Immunohistochemical analysis of Nectin-4 expression in esophageal squamous carcinoma specimens and adjacent normal tissues (n = 50). (B–D) Analysis of correlations between Nectin-4 expression and tumor grade, stage, age, and sex in ESCC patients, with statistical comparison between tumor and non-tumor tissues. (E) Immunohistochemical analysis of METTL3 expression in ESCC specimens and adjacent normal tissues (n = 50). (F–H) Analysis of correlations between METTL3 expression and tumor stage, grade, age, and sex in ESCC patients, with statistical comparison between tumor and non-tumor tissues. (I) Immunohistochemical analysis of VNN1 expression in ESCC tissues and adjacent normal tissues (n = 50). (K–L) Analysis of correlations between VNN1 expression and tumor grade, stage, age, and sex in ESCC patients, with statistical comparison between tumor and non-tumor tissues. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Immunohistochemical staining, Expressing, Comparison