CDC25A inhibition suppresses cell proliferation and induces G1/S‑phase cell cycle arrest in nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a primary malignancy that originates from the nasopharyngeal region. It has been demonstrated that a decrease in the expression level of cell division cycle gene 25A (CDC25A) suppresses cell viability and induces apoptosis in a variety of different types of cancer. However, at present, the role of CDC25A in NPC has yet to be fully elucidated. Therefore, the aim of the present study was to investigate the role of CDC25A in NPC progression and to explore the potential underlying mechanism. Reverse transcription‑quantitative PCR was performed to detect the relative mRNA levels of CDC25A and E2F transcription factor 1 (E2F1). Western blot analysis was subsequently used to determine the expression levels of CDC25A, Ki67, proliferating cell nuclear antigen (PCNA) and E2F1. CCK8 assay was employed to measure cell viability and flow cytometric analysis was employed to analyze the cell cycle. The binding sites between the CDC25A promoter and E2F1 were predicted using bioinformatics tools. Finally, luciferase reporter gene and chromatin immunoprecipitation assays were performed to verify the interaction between CDC25A and E2F1. The results obtained suggested that CDC25A is highly expressed in NPC cell lines and CDC25A silencing was found to inhibit cell proliferation, reduce the protein expression levels of Ki67 and PCNA and induce G1 arrest of NPC cells. Furthermore, E2F1 could bind CDC25A and positively regulate its expression at the transcriptional level. In addition, CDC25A silencing abolished the effects of E2F1 overexpression on cell proliferation and the cell cycle in NPC. Taken together, the findings of the present study showed that CDC25A silencing attenuated cell proliferation and induced cell cycle arrest in NPC and CDC25A was regulated by E2F1. Hence, CDC25A may be a promising therapeutic target for treatment of NPC.

Keywords: E2F transcription factor 1; cell cycle; cell division cycle gene 25A; nasopharyngeal carcinoma; proliferation.

Figure 1.

CDC25A was highly expressed in NPC cell lines. (A) The protein expression of CDC25A was determined by western blotting. (B) The relative mRNA expression of CDC25A was quantified by reverse transcription-quantitative PCR. The data were expressed as mean ± standard error of the mean of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. NP69. CDC25A, cell division cycle gene 25A; NPC, nasopharyngeal carcinoma.

Figure 2.

CDC25A silencing inhibited the proliferation of HK1 cells. (A) The protein expression of CDC25A was determined by western blotting. (B) The relative mRNA expression of CDC25A was quantified by reverse transcription-quantitative PCR. (C) HK1 cell viability was measured by CCK8 assay. (D) The expressions of Ki67 and PCNA were determined by western blotting. The data were expressed as mean ± standard error of the mean of three independent experiments. **P<0.01, ***P<0.001 vs. siRNA-NC. CDC25A, cell division cycle gene 25A; PCNA, proliferating cell nuclear antigen.

Figure 3.

CDC25A silencing induced G1 arrest of HK1 cells. (A) The cell cycle of HK1 cells was analyzed by flow cytometry and (B) quantified. The data were expressed as mean ± standard error of the mean with three independent times. (C) The protein expressions of Cyclin D1, CDK4 and CDK6 were determined by western blotting. **P<0.01, ***P<0.001 vs. siRNA-NC. CDC25A, cell division cycle gene 25A; CDK, cyclin-dependent kinase; si, short interfering; NC, negative control.

Figure 4.

CDC25A was transcriptionally regulated by E2F1 in HK1 cells. (A) The interaction between CDC25A and E2F1. (B) The search results from Cyclebase database. (C) The binding sites of CDC25A promoter and E2F1. The mutational fragments are in red. (D) The protein expression of E2F1 was determined by western blotting. (E) The relative mRNA expression of E2F1 was quantified by RT-qPCR. (F) The protein expression of CDC25A was determined by western blotting. (G) The relative mRNA expression of CDC25A was quantified by RT-qPCR. (H) The protein expression of E2F1 was determined by western blotting. (I) The relative mRNA expression of E2F1 was quantified by RT-qPCR. (J) The protein expression of CDC25A was determined by western blotting. (K) The relative mRNA expression of CDC25A was quantified by RT-qPCR. (L) The interaction between CDC25A and E2F1 was confirmed by luciferase reporter assay and ChIP assays. In luciferase reporter assay, FL group used the normal FL of CDC25A promoter while Site 1 and Site 2 groups used the FL of CDC25A promoter including the two mutational sequences of CDC25A promoter. (M) The interaction between CDC25A and E2F1 was confirmed by ChIP assay. The data were expressed as mean ± standard error of the mean of three independent experiments. ***P<0.001 vs. Control. CDC25A, cell division cycle gene 25A; E2F1, E2F transcription factor 1; RT-qPCR, reverse transcription-quantitative PCR; ChIP, chromatin immunoprecipitation; FL, full length.

Figure 5.

CDC25A silencing abolished the effect of E2F1 overexpression on cell proliferation and cell cycle in HK1 cells. (A) HK1 cell viability was measured by CCK8 assay. (B) The expression of Ki67 and PCNA were determined by western blot and (C) quantified. (D) The cell cycle of HK1 cells was analyzed by flow cytometry and (E) quantified. (F) The mRNA expressions of Cyclin D1, CDK4 and CDK6 were detected by qRT-PCR. The data were expressed as mean ± standard error of the mean of three independent experiments. ***P<0.001 vs. Control. ^###P<0.001 vs. OV-E2F1. CDC25A, cell division cycle gene 25A; E2F1, E2F transcription factor 1; PCNA, proliferating cell nuclear antigen; OV, overexpression.