DGKα Enhances Tumorigenic Activity in Bladder Cancer Patients With Chronic Kidney Disease

Figures

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  FIGURE 1. Generation of chronic kidney disease (CKD) model mice and Gene Set Enrichment Analysis (GSEA) of the bladder urothelium of CKD mice. (A) The elevation of serum blood urea nitrogen and creatinine in CKD model mice. (B) The upregulation of DGKα expression in the bladder urothelium of CKD mice compared with control mice. (C) Normalized enrichment scores of the top 10 upregulated pathways of hallmark gene sets enriched in the bladder urothelium of CKD mice compared with control mice. (D–F) Enrichment pattern of hallmark angiogenesis rerated (D), hallmark epithelial‐mesenchymal transition related (E), and G2M checkpoint‐related (F) gene sets determined by GSEA comparing CKD and control mice.

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  FIGURE 2. Relationship between DGKα expression and bladder cancer (BC) patients treated with radical cystectomy. (A) Immunohistochemical DGKα staining in BC patients. The results shown are representative of 42 independent specimens. Original magnification, ×100. Scale bar, 200 μm. (B) Immunohistochemical staining of DGKα‐positive and ‐negative tumors. The results shown are representative of 12 DGKα‐positive and 30 DGKα‐negative specimens. Original magnification, ×400. Scale bar, 50 μm. (C) Kaplan–Meier plots of recurrence‐free (left) and cancer‐specific (right) survival rates for DGKα‐positive and ‐negative BC patients. p‐values calculated by a log‐rank test are also indicated.

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  FIGURE 3. Successful knocking down of DGKα in BC cell lines. (A) DGKα‐knockdown (siDGKα‐1 and siDGKα‐2) BC cell lines were subjected to determine mRNA levels of DGKα by quantitative RT‐PCR analysis. Results are expressed as the mean ± SEM or representative of three experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by one‐way ANOVA combined with Dunnett's multiple comparisons test, compared with siCont. siCont, control siRNA‐introduced control. (B) DGKα‐knockdown (siDGKα‐1 and siDGKα‐2) BC cell lines were subjected to determine protein levels of DGKα by western blotting analysis.

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  FIGURE 4. Role of DGKα in BC cell invasion, migration, and proliferation in vitro. (A) DGKα‐knockdown cell lines were subjected to transwell invasion assay with corresponding control cells (siCont) (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001 by one‐way ANOVA combined with Dunnett's multiple comparisons test, compared with siCont. Representative Diff‐Quick‐stained transwell membranes using T24 cells were shown in left panels. Scale bar, 50 μm. (B) DGKα‐knockdown cell lines were subjected to a wound healing assay with corresponding control cells (siCtrl) (n = 3). *p < 0.05 and **p < 0.01 by one‐way ANOVA combined with Dunnett's multiple comparisons test, compared with siCont. Representative images of migrated cells using T24 cells were shown in the left panels. Scale bar, 50 μm. (C) DGKα‐knockdown cell lines were subjected to a cell proliferation assay with corresponding control cells (siCont) (n = 4). **p < 0.01 and ***p < 0.001 by one‐way ANOVA combined with Dunnett's multiple comparisons test, compared with siCont.