The inhibitory effect of silencing CDCA3 on migration and proliferation in bladder urothelial carcinoma

Abstract

Background: CDCA3 is an important component of the E3 ligase complex with SKP1 and CUL1, which could regulate the progress of cell mitosis. CDCA3 has been widely identified as a proto-oncogene in multiple human cancers, however, its role in promoting human bladder urothelial carcinoma has not been fully elucidated.

Methods: Bioinformatic methods were used to analyze the expression level of CDCA3 in human bladder urothelial carcinoma tissues and the relationship between its expression level and key clinical characteristics. In vitro studies were performed to validate the specific functions of CDCA3 in regulating cell proliferation, cell migration and cell cycle process. Alterations of related proteins was investigated by western blot assays. In vivo studies were constructed to validate whether silencing CDCA3 could inhibit the proliferation rate in mice model.

Results: Bioinformatic analysis revealed that CDCA3 was significantly up-regulated in bladder urothelial carcinoma samples and was related to key clinical characteristics, such as tumor grade and metastasis. Moreover, patients who had higher expression level of CDCA3 tend to show a shorter life span. In vitro studies revealed that silencing CDCA3 could impair the migration ability of tumor cells via down-regulating EMT-related proteins such as MMP9 and Vimentin and inhibit tumor cell growth via arresting cells in the G1 cell cycle phase through regulating cell cycle related proteins like p21. In vivo study confirmed that silencing CDCA3 could inhibit the proliferation of bladder urothelial carcinoma cells.

Conclusions: CDCA3 is an important oncogene that could strengthen the migration ability of bladder urothelial carcinoma cells and accelerate tumor cell growth via regulating cell cycle progress and is a potential biomarker of bladder urothelial carcinoma.

Keywords: Bladder urothelial carcinoma; CDCA3; Migration; cell cycle; p21.

Fig. 1

CDCA3 was related to crucial clinical characteristics and was a potential predictor of bladder urothelial carcinoma. a CDCA3 was significantly elevated in bladder urothelial carcinoma samples collected by the TCGA GDC database. b The expression of CDCA3 was higher in tumor tissues in the matched pairs. c Advanced bladder urothelial carcinoma samples showed a higher expression level of CDCA3. d Samples with distant metastasis presented a higher status of CDCA3. e CDCA3 was obviously higher in bladder urothelial carcinoma tissues than in normal bladder tissues, according to the GSE13507 microarray dataset. f The survival analysis of CDCA3 expression status with overall life span in bladder urothelial carcinoma patients. *P < 0.05, ***P < 0.001

Fig. 2

Six cell cycle genes correlated with CDCA3. a–f Correlation between CDCA3 and CDC25C (R = 0.78), BUB1 (R = 0.73), CCNB1 (R = 0.73), CDC20 (R = 0.78), CDC45 (R = 0.71) and PTTG1 (R = 0.71). g–i Survival analysis of the expression level of CCNB1, CDC20 and CDC25 in bladder urothelial carcinoma patients

Fig. 3

Silencing CDCA3 significantly inhibited bladder urothelial carcinoma cell proliferation in vitro. The efficiency of two CDCA3 specific siRNAs in 5637 cell line (a) and T24 cell line (b) by qRT-PCR. Western blot analysis of the siRNAs treatment in 5637 cell line (c) and T24 cell line (d). Cell viability tested by MTT assay in 5637 bladder urothelial carcinoma cells (e) and T24 bladder urothelial carcinoma cells (f). The effect of silencing CDCA3 on bladder urothelial carcinoma proliferation was tested by clone formation assay in 5637 cells (g, i) and T24 cells (h, j). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

The inhibitory effect of silencing CDCA3 on cell migration ability. Transwell migration assay performed in 5637 bladder urothelial carcinoma cells (a), in T24 bladder urothelial carcinoma cells (b) and statistically analyzed (c, d). The 24-h wound healing assay conducted in 5637 bladder urothelial carcinoma cells (e) and T24 bladder urothelial carcinoma cells (f) and the statistical analysis (g, h). Alteration of EMT-related proteins after CDCA3 specific siRNA transfection in 5637 cells (i) and T24 cells (j). Scale Bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Silencing CDCA3 arrested bladder urothelial carcinoma cells in G1 phase. Distributions of cell cycles detected by flow cytometry in 5636 cells (a) and T24 cells (b) after CDCA3 siRNA treatment and corresponding statistical analysis (c, d). Alteration of cell cycle related proteins responds to the silence of CDCA3 in 5637 bladder urothelial carcinoma cells (e) and T24 bladder urothelial carcinoma cells (f). *P < 0.05, **P < 0.01

Fig. 6

Silencing CDCA3 significantly inhibited bladder urothelial carcinoma cell proliferation in vivo. a The knockdown efficiency of lentiviral-CDCA3-shRNA in T24 cells. b Xenograft tumor volume was calculated at the 5th day, 14th day, 21st day, 28th day and 35th day after T24 cells were subcutaneously injected. c Lentivirus infected T24 cells were subcutaneously injected into BALB/C nude mice. 35 days later, the mice were sacrificed and xenograft tumors were dissected. d Weight of the xenograft tumors was measured after mice were sacrificed. e Representative IHC images of xenograft tissues from the tumor-bearing mice. Scale bar: 20 μm. *P < 0.05, **P < 0.01