Journal: Genes & Diseases

Article Title: Menin facilitates the cell proliferation of bladder cancer via modulating the TFAP2C/β-catenin axis

doi: 10.1016/j.gendis.2025.101565

Figure Lengend Snippet: The MEN1 gene is identified as an oncogene in BLCA. (A) The expression status of the MEN1 gene in BLCA datasets based on the TCGA database. (B) The correlation between the MEN1 gene and tumor stages in BLCA tissues based on the TCGA database. (C) The survival analysis indicates that the MEN1 gene is a risk factor and is related to the survival and prognosis of BLCA tissues based on the GEO database ( GSE31684 and GSE13507 ). (D) Representative immunohistochemistry staining images are presented for immunostaining of menin in different stages of BLCA tissues. Scale bar, 100 μm. (E) Enrichment analysis of the high expression of the MEN1 gene of BLCA patients based on GSEA 4.2.3 software ( https://www.gsea-msigdb.org/gsea/index.jsp ). (F) The correlation between the MEN1 gene and cell cycle checkpoint-related markers in BLCA based on the TCGA database. (G, H) The quantitative real-time PCR and western blotting assays analyzed the expression level of the MEN1 gene and menin protein in human immortalized uroepithelial cell line (SV-HUC-1) and BLCA cell lines (T24, 5637, and HT-1197), respectively. (I) Images of representative nude mouse xenograft model studies. T24 tumor xenografts excised from male BALB/c (nu/nu) nude mice after 28 days of treatments with the in vivo RNA interfering of MEN1 knockdown (MEN1-KD group) or control. (J) Days versus tumor volume curves for T24 tumor xenografts showed that MEN1-KD inhibited tumor growth compared with the control group. (K) Representative immunohistochemistry staining images of menin, Ki67, CDK2, and CDK4 were presented in MEN1-KD-treated or control-treated T24 xenograft nude mice tissues. Scale bar, 50 μm ns, non-significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.001 versus control. MEN1, multiple endocrine neoplasia type 1; BLCA, bladder cancer; CDK2/4, cyclin-dependent kinase 2/4.

Article Snippet: BLCA cell lines, including human immortalized uroepithelial cells (SV-HUC-1) and three BLCA cell lines (T24, 5637, and HT-1197), were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunohistochemistry, Staining, Immunostaining, Software, Real-time Polymerase Chain Reaction, Western Blot, In Vivo, Knockdown, Control

Journal: Genes & Diseases

Article Title: Menin facilitates the cell proliferation of bladder cancer via modulating the TFAP2C/β-catenin axis

doi: 10.1016/j.gendis.2025.101565

Figure Lengend Snippet: Reduced menin inhibits BLCA cell proliferation via modulating cell cycle transition. (A) RT-qPCR analysis of MEN1 transcription in siCtrl-, siMEN1#1-, or siMEN1#2-treated T24, 5637, and HT-1197 BLCA cells. Data were normalized against ACTB and represented as fold change. (B) Western blotting analysis showed menin expression in siCtrl-, siMEN1#1-, or siMEN1#2-treated T24, 5637, and HT-1197 BLCA cells. (C) CCK-8 analysis showed the effect of MEN1-KD (siMEN1#1, siMEN1#2) on the cell proliferation of the tested BLCA cell lines. (D) Representative images of colony formation assays and their quantification in MEN1-KD T24, 5637, and HT-1197 cells. (E) MEN1-KD induces cell cycle arrest at the G1/S phase in T24, 5637, and HT-1197 cells, compared with the cells transfected with siCtrl-by flow cytometry. Percentages (%) of cell populations at different stages of cell cycles are listed within the panels. (F, G) The relative expression of MEN1 , CDK2 , CDK4 , CCND1 , and CCNE1 were measured in MEN1-KD T24, 5637, and HT-1197 cells by RT-qPCR or western blotting analysis. The experiment was repeated three times, and representative results are presented. ns, non-significant; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus control. BLCA, bladder cancer; RT-qPCR, quantitative real-time PCR; MEN1, multiple endocrine neoplasia type 1; CDK2/4, cyclin dependent kinase 2/4; CCND1, cyclin D1; CCNE1, cyclin E1.

Article Snippet: BLCA cell lines, including human immortalized uroepithelial cells (SV-HUC-1) and three BLCA cell lines (T24, 5637, and HT-1197), were purchased from the American Type Culture Collection (ATCC).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Transfection, Flow Cytometry, Control, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Menin facilitates the cell proliferation of bladder cancer via modulating the TFAP2C/β-catenin axis

doi: 10.1016/j.gendis.2025.101565

Figure Lengend Snippet: Menin regulates TFAP2C transcription to promote BLCA cell growth. (A) Heatmap of transcriptional changes triggered by negative control or MEN1-KD based on RNA sequencing data of T24 cells transfected with siCtrl or siMEN1. (B, C) mRNA and protein expression of TFAP2C was analyzed by RT-qPCR and western blotting in TFAP2C-KD T24, 5637, and HT-1197 cells. (D) CCK-8 analysis showed the effect of TFAP2C-KD (siTFAP2C#1, siTFAP2C#2) on the cell proliferation of the tested BLCA cell lines. (E) Representative images of colony formation assays and their quantification in TFAP2C-KD T24, 5637, and HT-1197 cells. (F) RT-qPCR analysis showed the down-regulated TFAP2C expression in MEN1-KD T24, 5637, and HT-1197 cells. (G) Western blotting analysis showed MEN1-KD caused a strong reduction of TFAP2C expression in T24, 5637, and HT-1197 cells. (H) Representative immunohistochemistry images were presented for the immunostaining of menin and TFAP2C in MEN1-KD-treated or negative control-treated T24 xenograft nude mouse tissues. (I) Schematic representation of the position of ChIP-qPCR primers along the TFAP2C promoter. (J – M) The levels of menin, H3K4me3, MLL1, or ASH2L recruitment to the TFAP2C promoter in siCtrl- or siMEN1#1 + #2-transfected T24 cells, detected by ChIP-qPCR. Scale bar, 50 μm ns, non-significant; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus control. TFAP2C, transcription factor AP-2 gamma; BLCA, bladder cancer; RT-qPCR, quantitative real-time PCR; MEN1, multiple endocrine neoplasia type 1; ChIP, chromatin immunoprecipitation; H3K4me3, histone 3 lysine 4 trimethylation; MLL1, mixed lineage leukemia 1.

Article Snippet: BLCA cell lines, including human immortalized uroepithelial cells (SV-HUC-1) and three BLCA cell lines (T24, 5637, and HT-1197), were purchased from the American Type Culture Collection (ATCC).

Techniques: Negative Control, RNA Sequencing, Transfection, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Immunohistochemistry, Immunostaining, ChIP-qPCR, Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Genes & Diseases

Article Title: Menin facilitates the cell proliferation of bladder cancer via modulating the TFAP2C/β-catenin axis

doi: 10.1016/j.gendis.2025.101565

Figure Lengend Snippet: MEN1 silencing reduces cell growth via suppressing Wnt/β-catenin signaling in BLCA cells. (A) T24 cells transfected by siCtrl or siMEN1-#1 + #2 were harvested for transcriptomic RNA sequencing. The volcano plot depicts the gene expression changes in siMEN1-treated T24 cells. (B) KEGG pathway enrichment analysis based on RNA sequencing showed the pathway enrichment of differentially expressed genes. The bubble size indicates the number of genes. The color bar indicates the corrected p -value. The threshold for differential was set at 2-fold change, and p < 0.05, as determined by DESeq2. (C) RT-qPCR analysis showed the down-regulated CTNNB1 expression in MEN1-KD T24, 5637, and HT-1197 cells. (D) The protein expression of β-catenin was analyzed by western blotting in MEN1-KD T24, 5637, and HT-1197 cells, as indicated. (E) Schematic representation of the position of ChIP-qPCR primers along the CTNNB1 promoter. (F) The levels of menin recruitment to the CTNNB1 promoter in siCtrl- or siMEN1#1 + #2-transfected BLCA cells, detected by ChIP-qPCR assay. (G) The β-catenin recruitment on the CDK2 , CDK4 , CCNE1 , CCND1 , and MYC promoter in siCtrl- or siMEN1#1 + #2-transfected BLCA cells, detected by ChIP-qPCR. ns, non-significant; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus control. BLCA, bladder cancer; RT-qPCR, quantitative real-time PCR; MEN1, multiple endocrine neoplasia type 1; CTNNB1, catenin beta 1; ChIP, chromatin immunoprecipitation; CDK2/4, cyclin dependent kinase 2/4; CCND1, cyclin D1; CCNE1, cyclin E1.

Article Snippet: BLCA cell lines, including human immortalized uroepithelial cells (SV-HUC-1) and three BLCA cell lines (T24, 5637, and HT-1197), were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Expressing, Western Blot, ChIP-qPCR, Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Genes & Diseases

Article Title: Menin facilitates the cell proliferation of bladder cancer via modulating the TFAP2C/β-catenin axis

doi: 10.1016/j.gendis.2025.101565

Figure Lengend Snippet: TFAP2C is crucial for the regulation of CTNNB1 by menin in BLCA cells. (A, B) mRNA and protein expression of TFAP2C , CTNNB1 , CDK2 , CDK4 , CCND1 , CCNE1 , and MYC were analyzed by RT-qPCR and western blotting in TFAP2C-KD T24, 5637, and HT-1197 cells. (C) Schematic representation of the position of ChIP-qPCR primers along the CTNNB1 promoter. (D) The levels of TFAP2C recruitment to the CTNNB1 promoter in siCtrl or siTFAP2C-treated T24, 5637, and HT-1197 cells, detected by ChIP-qPCR. (E) RT-qPCR analysis showed the expression of MEN1 , TFAP2C , and CTNNB1 in OE-menin-treated, siTFAP2C-treated, or OE-menin plus siTFAP2C-treated BLCA cells. (F) The levels of menin recruitment to the CTNNB1 promoter in OE-menin-treated, or OE-menin plus siTFAP2C-treated T24, 5637, and HT-1197 cells, detected by ChIP-qPCR. ns, non-significant; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus control. TFAP2C, transcription factor AP-2 gamma; BLCA, bladder cancer; RT-qPCR, quantitative real-time PCR; MEN1, multiple endocrine neoplasia type 1; CTNNB1, catenin beta 1; ChIP, chromatin immunoprecipitation; CDK2/4, cyclin dependent kinase 2/4; CCND1, cyclin D1; CCNE1, cyclin E1.

Article Snippet: BLCA cell lines, including human immortalized uroepithelial cells (SV-HUC-1) and three BLCA cell lines (T24, 5637, and HT-1197), were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, ChIP-qPCR, Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation