https://orcid.org/0000-0001-6577-5403
Figures & data
Figure 1. CRTAC1 is downregulated in bladder cancer tissues and cells.
(a-b) CRTAC1 expression in tissue samples of bladder cancer was predicted by the GEPIA and starBase websites. (c) The survival analysis of bladder cancer patients with high or low expression of CRTAC1 was analyzed by Kaplan–Meier Plotter. (d) CRTAC1 level in bladder cancer tissue samples (n = 20) was detected by RT-qPCR. (e) The mRNA and protein expression of CRTAC1 in bladder cancer cell lines (T24, 5637) and normal urothelial SVHUC-1 cells was examined by RT-qPCR. (F) Immunofluorescence staining was used to assess the expression and location of CRTAC1 expression in bladder cancer cell lines and normal urothelial cell line. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2. CRTAC1 inhibits bladder cancer cell proliferation, migration, invasion and EMT process.
(a-b) RT-qPCR was used to assess the overexpression or knockdown efficiency of CRTAC1 in T24 and 5637 cells respectively. (c) The viability of T24 and 5637 cells after CRTAC1 overexpression or knockdown was detected using CCK-8 assays. (d) The proliferative ability of T24 and 5637 cells after indicated transfection was detected using colony formation assays. (e) Wound healing assays were conducted to assess the migratory ability of T24 and 5637 cells after CRTAC1 overexpression or knockdown. (f) Transwell assays were performed to detect the invasion of T24 and 5637 cells overexpressing or silencing CRTAC1 expression. (g) Protein levels of EMT markers (E-cadherin, N-cadherin, Slug and Twist) were detected by western blotting in T24 and 5637 cells after the above transfection. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3. CRTAC1 targets YY1 in bladder cancer cells.
(a) Pearson correlation analysis was used to assess the expression correlation between CRTAC1 and YY1 in bladder cancer tissues. (b) The immunofluorescent staining was used to detect the expression and colocalization of CRTAC1 and YY1 in the nucleus of bladder cancer cells. (c) The interaction between CRTAC1 and YY1 was explored by ChIP and Co-IP assays. (d) Luciferase reporter assay was used to explore the binding between CRTAC1 and YY1 promoter. (e) YY1 expression in bladder cancer tissue samples was examined by RT-qPCR. (f) The mRNA and protein levels of YY1 in T24, 5637 and SVHUC-1 cells were examined by RT-qPCR and western blotting. (g) YY1 expression in bladder cancer cells after CRTAC1 overexpression or silencing. **p < 0.01, ***p < 0.001.
Figure 4. CRTAC1 inactivates the TGF-β signaling pathway by downregulating YY1.
(a) The overexpression efficiency of YY1 in T24 cells was detected using RT-qPCR. (b-c) The protein expression of TGF-β, p-SMAD2/3, SMAD2/3, p-SMAD1 and SMAD1 in T24 cells after CRTAC1 overexpression and YY1 overexpression was detected by western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5. CRTAC1 inhibits malignant behaviors of bladder cancer cells by downregulating YY1.
(a-b) The expression levels of CRTAC1 and YY1 in T24 cells after transfection of pcDNA3.1/CRTAC1, empty pcDNA3.1 vector or co-transfection of pcDNA3.1/CRTAC1 and pcDNA3.1/YY1 were examined by RT-qPCR. (c-d) CCK-8 and colony formation assays were performed to measure the viability and proliferation of T24 cells after the above transfection. (e-f) The migration and invasion of T24 cells after CRTAC1 and YY1 overexpression was assessed by wound healing and Transwell assays. (g) The protein levels EMT markers in T24 cells after the above transfection were detected by western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.
Data availability statement
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.