Dysregulation of the cell cycle contributes to tumor progression. Cell division cycle-associated 3 (CDCA3) is a known trigger of mitotic entry and has been demonstrated to be constitutively upregulated in tumors. It is therefore associated with carcinogenic properties reported in various cancers. However, the role of CDCA3 in prostate cancer is unclear. In the present study, western blotting and analysis of gene expression profiling datasets determined that CDCA3 expression was upregulated in prostate cancer and was associated with a poor prognosis. CDCA3 knockdown in DU145 and PC-3 cells led to decreased cell proliferation and increased apoptosis, with increased protein expression levels of cleaved-caspase3. Further experiments demonstrated that downregulated CDCA3 expression levels induced G0/G1 phase arrest, which was attributed to increased p21 protein expression levels and decreased cyclin D1 expression levels via the regulation of NF-κB signaling proteins (NFκB-p105/p50, IKKα/β, and pho-NFκB-p65). In conclusion, these results indicated that CDCA3 may serve a crucial role in prostate cancer and consequently, CDCA3 knockdown may be used as a potential therapeutic target.
Prostate cancer is the most common type of malignant tumor in males and is the leading cause of cancer-related mortality in numerous countries (
Generally, the prognosis of patients with prostate cancer depends on the depth of tumor infiltration, as well as the appearance of lymph nodes and long-distance metastases, which can be assessed by pathological microscopy (
Currently, researchers are focused on targeting the cell cycle to exploit and block the proliferative capacity of cancer cells. Progression through the phases of the cell cycle relies on a complex network of proteins. Previous studies have suggested that the abnormal expression of cell cycle regulatory proteins may contribute to the development of cancer (
The aim of the present study was to determine, using microarray analysis, whether CDCA3 served as a hub gene in prostate cancer progression and was associated with patient prognosis. It was also investigated whether CDCA3 was differentially expressed in prostate cancer and paracancerous tissues and if CDCA3 was essential for cell proliferation, apoptosis and cell cycle arrest.
Gene expression profiling datasets for prostate cancer were analyzed using the Gene Expression Omnibus (GEO;
UALCAN (
Search Tool for the Retrieval of Interacting Genes (STRING) is a biological database (
Disease-free survival (DFS) of patients with prostate cancer was analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA;
The present study was approved (approval no. xs2020ky014) by the Ethics Committee of Xishan People's Hospital of Wuxi City (Wuxi, China). In total, seven prostate cancer samples were collected to investigate CDCA3 protein expression. Both cancerous and paracancerous tissue samples were collected from each patient. All patients (mean age, 74 years; range, 68–79 years) were treated with radical prostatectomy between January 2021 and July 2021, and consents were obtained orally. The inclusion criteria were tissue samples collected from patients who had not undergone androgen deprivation therapy, chemotherapy, radiotherapy, or other auxiliary treatment prior to surgery. The exclusion criteria were tissue samples collected from patients who had other severe comorbidities.
The human prostate cancer DU145 (serial cat. no. TCHu222) and PC-3 (serial cat. no. TCHu158) cell lines were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. CDCA3 protein levels were highly expressed in DU145 and PC-3 cell lines as revealed by analyzing the Harmonizome (
DU145 and PC-3 cells were seeded at 200,000 cells/well into 6-well plates and cultured in RPMI-1640 medium containing 10% FBS at 37°C with 5% CO2 overnight for transfection. Prostate cancer cells were transduced with sh-CDCA3 or sh-NC at a multiplicity of infection of 50 and cultured with 6 µg/ml polybrene (GeneChem Co. Ltd) for 12 h in a humidified atmosphere at 37°C with 5% CO2. Subsequently, the cells were seeded at 2,000 cells/well in a 96-well plate. At 24–96 h post-transfection, cell viability was detected using a Cell Counting Kit-8 (CCK-8) assay kit (Beyotime Institute of Biotechnology). Cells were incubated with 10 µl CCK-8 solution/well for 1 h at 37°C according to the manufacturer's protocol. The absorbance was measured at a wavelength of 450 nm. Cell viability was measured across five wells in each group. All independent treatments were carried out in three replicates.
DU145 and PC-3 cells in the logarithmic growth phase were collected and 1,000 cells/well were seeded into a 6-well plate. A total of 3 replicate wells were used for each group. Cells were cultured in RPMI-1640 medium containing 10% FBS at 37°C with 5% CO2 for 10 days and when macroscopic colonies appeared the culture solution was discarded. After washing with PBS, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature and stained with 0.5% crystal violet solution for 15 min at room temperature. Colonies were observed using a light microscope and the number of colonies was counted by visual inspection. Images were captured using a Canon EOS600D digital camera (Canon, Inc.). The minimum number of cells per colony was 50.
The apoptotic rate and cell cycle of prostate cancer cells were investigated using flow cytometry. DU145 and PC-3 cells were trypsinized (without EDTA). For the cell cycle analysis, cells were washed with PBS and subsequently incubated for 30 min at 37°C in the dark with 500 µl PI (Beyotime Institute of Biotechnology). Cells were then scanned using a CytoFLEX flow cytometer (Beckman Coulter, Inc.). Cells were counted and the percentages of prostate cancer cells in the three cycle phases were compared. For the cell apoptosis analysis, cells were washed with PBS and were subsequently cultured for 30 min at 37°C after adding 5 µl Annexin V-phycoerythrin and 10 µl 7-aminoactinomycin D (Hangzhou Multi Sciences Biotech Co., Ltd.) to identify apoptotic and necroptotic cells. Stained cells and their apoptotic rates were quantified using the software CytExpert 2.4.0.28 (Beckman Coulter, Inc.).
Total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined using a BCA assay (Beyotime Institute of Biotechnology). Total protein (30 µg/lane) was separated using SDS-PAGE on a 6–12% gel (Beyotime Institute of Biotechnology) and transferred onto a PVDF membrane (MilliporeSigma). The membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated overnight at 4°C with diluted primary antibodies. Subsequently, the membranes were washed using TBS with 0.1% Tween-20 (TBST) three times and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After being washed with TBST, the membranes were visualized using electrochemiluminescence (ECL) kit (Beyotime Institute of Biotechnology). GAPDH was used as the loading control. The following antibodies were used: Mouse monoclonal anti-GAPDH (1:2,000; cat. no. 33033M; BIOSS), rabbit polyclonal anti-CDCA3 (1:1,000; cat. no. YT0819), rabbit polyclonal anti-cleaved caspase-3 (1:1,000; cat. no. YC0006), rabbit polyclonal anti-pro-caspase-3 (1:1,000; cat. no. YT6113), and rabbit polyclonal anti-cyclin-dependent kinase inhibitor 1 (p21; 1:1,000; cat. no. YT3497; all from Immunoway Biotechnology Company), rabbit polyclonal anti-cyclin D1 (1:1,000; cat. no. 0623R; BIOSS), mouse monoclonal anti-NFκB-p65 (1:1,000; cat. no. YM311), rabbit polyclonal anti-phosphorylated (p)-NFκB-p65 (1:1,000; cat. no. YP0192), rabbit polyclonal anti-IKKα/β (1:1,000; cat. no. YT2302), and rabbit polyclonal anti-NFκB-p105/p50 (1:1,000; cat. no. YT3101; all from Immunoway Biotechnology Company), HRP-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. 40295G-HRP; BIOSS) and HRP-labeled goat anti-mouse secondary antibody (1:5,000; cat. no. 0368G-HRP; BIOSS).
The softwares SPSS 17.0 (SPSS, Inc.) and ImageJ 1.8.0 (National Institutes of Health) were used to carry out statistical analysis. Each value was acquired from at least three independent experiments. Data are presented as the mean ± SD. A two-tailed unpaired Student's t-test was used to analyze statistical differences between two groups. P<0.05 was considered to indicate a statistically significant difference.
In total, 1,119 and 1,690 DEGs were identified via the analysis of normal prostate vs. cancer tissues in the GSE27616 and GSE3325 datasets, respectively. A comparison of these two sets of genes revealed 231 overlapping genes, including 60 upregulated and 171 downregulated DEGs (
CDCA3 mRNA was demonstrated to be upregulated in prostate cancer compared with normal prostate tissues, both in GSE27616 and GSE3325 datasets. These results were also supported by TCGA database. In total, 497 prostate cancer tissues and 52 normal prostate samples were identified. CDCA3 was determined to be significantly upregulated in prostate cancer compared with normal tissues (median 2.372 vs. 0.856 transcripts per million; P<0.001;
To verify the aforementioned results, prostate cancer and paracancerous tissues were collected from seven patients with prostate cancer at the Xishan People's Hospital of Wuxi city. Proteins were extracted from the tissues and the CDCA3 protein expression level was examined. The western blotting results revealed that CDCA3 protein expression levels were significantly higher in the prostate cancer tissue samples compared with the paracancerous tissue samples (P<0.01;
Western blotting was used to detect the inhibition of CDCA3 protein expression following transduction with sh-CDCA3. CDCA3 protein expression levels were suppressed by 61.9% in DU145 cells and 66.3% in PC-3 cells transduced with sh-CDCA3 compared with the sh-NC transduced cells (P<0.001;
Subsequently, the effect of CDCA3 knockdown on DU145 and PC-3 cell proliferation was investigated using the CCK-8 and colony formation assays. The CCK-8 assay results demonstrated that knockdown of CDCA3 inhibited cell viability by 2.7% after 48 h (P=0.28), 26.2% after 72 h (P<0.001), 29.7% after 96 h (P<0.001) in DU145 cells, and 13.7% after 48 h (P<0.001), 37.0% after 72 h (P<0.001) and 34.7% after 96 h (P<0.001) in PC-3 cells, compared with the sh-NC cell group (
The mechanism of CDCA3 in cell apoptosis regulation was assessed using flow cytometry. The results demonstrated that CDCA3 knockdown promoted early apoptosis both in DU145 (45.3% in sh-CDCA3 vs. 5.1% in sh-NC; P<0.001) and PC-3 cells (37.8% in sh-CDCA3 vs. 4.9% in sh-NC; P<0.001). The late apoptotic rate was also increased in DU145 (7.1% in sh-CDCA3 vs. 5.3% in sh-NC; P<0.005) and PC-3 cells (13.8% in sh-CDCA3 vs. 8.9% in sh-NC; P<0.005;
The role of CDCA3 in modulating the prostate cancer cell cycle was analyzed using flow cytometry. The results demonstrated that CDCA3 knockdown induced G0/G1 phase arrest. The percentage of cells in the G0/G1 phase in the sh-CDCA3 group was higher compared with the sh-NC group, both in DU145 (35.06% in sh-CDCA3 vs. 30.91% in sh-NC; P<0.001) and PC-3 cells (36.61% in sh-CDCA3 vs. 33.06% in sh-NC; P<0.01;
Cyclin D1, a regulator of G1 phase progression, serves a crucial role in carcinogenesis and cancer progression (
NF-κB activation is related to tumor initiation, progression and metastasis in prostate cancer (
To discover the role of CDCA3 knockdown in the regulation of the NF-κB signaling pathway, western blotting was performed. The results demonstrated that CDCA3 knockdown suppressed the protein expression levels of NFκB-p105/p50, IKKα/β and p-NFκB-p65, both in DU145 and PC-3 cells (
Abnormal cell division can lead to cancer. Disturbance of cell cycle regulation is an important biological feature exhibited in malignant tumors and can lead to reduced apoptosis, unlimited proliferation and metastasis in malignant cells (
Previous studies have determined that CDCA3 expression levels are increased in tumor tissues and associated with a poor patient prognosis in CRC, GC, NSCLC, OSCC and PAC (
Using the GEO database, the present study identified 20 hub genes that were expressed at elevated levels in prostate cancer compared with normal prostate tissues. The transcriptomic levels of CDCA3 were also significantly upregulated in prostate cancer compared with normal tissues in TCGA database. It was further verified by western blotting that CDCA3 protein expression levels were upregulated in prostate cancer. Using the GEPIA database, it was determined that high CDCA3 expression levels were associated with a poor prognosis, as discussed for the numerous other aforementioned tumors. The results also demonstrated that knockdown of CDCA3 in DU145 and PC-3 cells inhibited cell proliferation and facilitated early and late apoptosis with an increase in the protein expression levels of cleaved caspase-3.
Regarding the molecular mechanism of CDCA3 in the cell cycle, the NF-κB signaling pathway was further investigated. NF-κB is a major regulator of numerous important cell processes, such as inflammation, proliferation and apoptosis (
However, there are still many topics that remain to be explored. Firstly, the effects of CDCA3 on the NF-κB signaling pathway and the mechanisms involved need further investigation. RNA-seq analysis will be performed to identify DEGs between sh-CDCA3 and sh-NC in response to CDCA3 knockdown in DU145 and PC-3 cell lines, and the mechanisms of CDCA3 interacting with DEGs will be investigated. Secondly, to further confirm the effects of CDCA3
In conclusion, these data demonstrated that CDCA3 was upregulated in prostate cancer tissues and was associated with a poor prognosis. The results indicated that knockdown of CDCA3 potentially suppresses prostate cancer progression via the significant accumulation of p21 and via inhibiting the expression of cyclin D1 by regulating the NF-κB signaling pathway.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
DY, XH, and PG designed the study. PG and MZ carried out the study, including data collection and data analysis. JZ performed data analysis. PG wrote the manuscript. PG and MZ confirm the authenticity of the raw data. All the authors have read and approved the final manuscript.
The present study was approved (approval no. xs2020ky014) by the Ethics Committee of Xishan People's Hospital of Wuxi City (Wuxi, China).
Not applicable.
The authors declare that they have no competing interests.
Hub gene screening and survival analysis. (A) In total, 257 and 1,060 DEGs were upregulated in prostate cancer tissues in the GSE27616 and GSE3325 datasets, respectively, and 60 overlapping genes were identified. (B) In total, 862 and 630 DEGs were downregulated in prostate cancer tissues in the GSE27616 and GSE3325 datasets, respectively, and 171 overlapping genes were identified. (C) A protein-protein interaction network of hub genes was constructed using Cytoscape. (D) Disease-free survival analysis of CDCA3 in prostate cancer patients was performed using the Gene Expression Profiling Interactive Analysis database. DEG, differentially expressed gene; CDCA3, cell division cycle-associated 3.
CDCA3 overexpression in prostate cancer tissues. (A) Transcriptomic levels of CDCA3 in prostate cancer and normal prostate tissues (The Cancer Genome Atlas database). (B) Western blotting was performed to determine CDCA3 protein expression levels in seven prostate cancer tissue samples compared with paracancerous tissue samples. The serial number of each case is displayed at the top of the figure. Data are presented as the mean ± SD. **P<0.01 and ***P<0.001. CDCA3, cell division cycle-associated 3; C, cancer tissue; P, paracancerous tissue.
CDCA3 protein expression levels were suppressed in DU145 and PC-3 cells infected with sh-CDCA3 lentivirus. Protein expression levels of apoptosis-associated proteins, including pro-caspase-3 and cleaved caspase-3, were analyzed via western blotting. GAPDH was used as a loading control. Data are presented as the mean ± SD of three independent experiments. ***P<0.001. CDCA3, cell division cycle-associated 3; sh-, short hairpin; NC, negative control.
Knockdown of CDCA3 inhibits DU145 and PC-3 cell progression. (A) Effects of CDCA3 on cell proliferation were determined using the Cell Counting Kit-8 assay. Data are presented as the mean ± SD of five samples. (B) Effects of CDCA3 on cell proliferation were determined using the colony formation assay. (C) Flow cytometry determined that the downregulation of CDCA3 promoted early and late apoptosis. Data are presented as the mean ± SD of three samples. **P<0.01 and ***P<0.001. CDCA3, cell division cycle-associated 3; sh-, short hairpin; NC, negative control.
Knockdown of CDCA3 induces G0/G1-phase arrest in DU145 and PC-3 cells. (A) Flow cytometry was used to analyze the cell cycle following CDCA3 knockdown in DU145 and PC-3 cells. (B) Cell cycle-associated protein levels, including cyclin D1 and p21, were analyzed via western blotting. GAPDH was used as a loading control. Data are presented as the mean ± SD of three independent experiments. **P<0.01 and ***P<0.001. CDCA3, cell division cycle-associated 3; sh-, short hairpin; NC, negative control.
CDCA3 knockdown exerts an inhibitory effect on the NF-κB signaling pathway. Protein expression levels of NFκB-p105/p50, IKKα/β, NFκB-p65 and p-NFκB-p65 were determined via western blotting. GAPDH was used as the loading control. Data are presented as the mean ± SD of three independent experiments. **P<0.01 and ***P<0.001. CDCA3, cell division cycle-associated 3; p-, phosphorylated; sh-, short hairpin; NC, negative control.