Journal: Journal of Translational Medicine

Article Title: Glutamine metabolism reprogramming promotes bladder cancer progression via PYCR1: a multi-omics and functional validation study

doi: 10.1186/s12967-025-07386-2

Figure Lengend Snippet: Glutamine metabolism profiling and single-cell landscape in bladder cancer. ( A ) Schematic of targeted metabolomics workflow comparing BLCA tumor cells and normal urothelial cells. ( B ) Boxplot showing differential amino acid expression between tumor and normal cell lines. ( C ) UMAP clustering of single-cell transcriptomes from BLCA tissue samples. ( D ) Cell type annotation based on canonical markers. ( E ) Differential gene expression analysis (DEGs) across cell clusters. ( F ) Glutamine metabolism module score visualized by UMAP. ( G ) Urothelial cells colored by high/low glutamine score group. ( H ) Volcano plot of DEGs between high- and low-score groups. ( I ) GO enrichment analysis of DEGs showing involvement in metabolic and apoptotic pathways. Error bars indicate mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human BLCA cell lines (T24, J82, UMUC3, SW780) and a normal urothelial cell line (SV-HUC-1) were obtained from American Type Culture Collection (ATCC).

Techniques: Expressing, Gene Expression

Journal: Journal of Translational Medicine

Article Title: Glutamine metabolism reprogramming promotes bladder cancer progression via PYCR1: a multi-omics and functional validation study

doi: 10.1186/s12967-025-07386-2

Figure Lengend Snippet: Knockdown or inhibition of PYCR1 suppresses proliferation, migration, and tumor growth of BLCA cells in vitro and in vivo. ( A ) Western blot showing PYCR1 expression levels in BLCA cell lines and normal urothelial cell line SV-HUC-1. ( B ) Validation of PYCR1 knockdown efficiency in T24 and J82 cells using three different shRNAs. ( C ) CCK-8 assay showing reduced proliferation of T24 and J82 cells upon PYCR1 knockdown. ( D ) Colony formation assay indicating a significant decrease in clonogenic ability after PYCR1 knockdown. ( E ) Transwell migration assay demonstrating impaired migration in PYCR1 knockdown cells, scale bar = 200 μm. ( F ) Flow cytometry analysis of cell cycle distribution in J82 and T24 cells after PYCR1 knockdown. The G2 phase showed significant accumulation with P = 0.0036 for J82 cells and P = 0.0055 for T24 cells. ( G ) Flow cytometry analysis of apoptosis in T24 and J82 cells following PYCR1 knockdown. ( H ) Schematic diagram of xenograft mouse models used to evaluate the effects of PYCR1 inhibition in vivo ( n = 6 mice per group). ( I ) Tumor volume comparison and IHC staining (H&E, PYCR1, Ki-67) of xenografts from T24 cells treated with PYCR1 inhibitor versus DMSO control. ( J ) Tumor volume comparison and IHC staining of xenografts from T24-NC and T24-shPYCR1 cells. Data are presented as mean ± SD from three independent experiments. ns indicates P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Human BLCA cell lines (T24, J82, UMUC3, SW780) and a normal urothelial cell line (SV-HUC-1) were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Inhibition, Migration, In Vitro, In Vivo, Western Blot, Expressing, Biomarker Discovery, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Flow Cytometry, Comparison, Immunohistochemistry, Control

Journal: Journal of Translational Medicine

Article Title: Glutamine metabolism reprogramming promotes bladder cancer progression via PYCR1: a multi-omics and functional validation study

doi: 10.1186/s12967-025-07386-2

Figure Lengend Snippet: PYCR1 knockdown impairs proline synthesis and suppresses the PI3K/AKT/mTOR signaling pathway in BLCA cells. ( A ) Quantification of intracellular proline levels in T24 and J82 cells after PYCR1 knockdown. ( B ) Correlation analysis showing positive associations between PYCR1 expression and SLC1A5 (left) and P5CS (right) in BLCA samples. ( C ) Western blot analysis confirming that knockdown of PYCR1 reduces protein expression levels of P5CS and SLC1A5. ( D ) Heatmap of differentially expressed genes in BLCA cells following PYCR1 knockdown from RNA-seq data. ( E - F ) Pathway enrichment analyses of downregulated ( E ) and upregulated ( F ) genes after PYCR1 knockdown, indicating involvement in PI3K-AKT and immune-related signaling pathways. ( G ) Western blot analysis validating the downregulation of PI3K, AKT, and mTOR pathway components upon PYCR1 knockdown in T24 and J82 cells. ( H ) Western blot analysis of PI3K/AKT/mTOR pathway activation following PYCR1 overexpression with or without LY294002 treatment in T24 and J82 cells. PYCR1 overexpression increased phosphorylation of PI3K, AKT, and mTOR, which was reversed by the PI3K inhibitor. Vinculin served as the loading control. Data are presented as mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01; oe: Overexpression

Article Snippet: Human BLCA cell lines (T24, J82, UMUC3, SW780) and a normal urothelial cell line (SV-HUC-1) were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Expressing, Western Blot, RNA Sequencing, Protein-Protein interactions, Activation Assay, Over Expression, Phospho-proteomics, 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: 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