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Epigenetic Mediated Zinc Finger Protein 671 Downregulation Promotes Cell Proliferation and Tumorigenicity in Nasopharyngeal Carcinoma by Inhibiting Cell Cycle Arrest

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Epigenetic Mediated Zinc Finger Protein 671 Downregulation Promotes Cell Proliferation and Tumorigenicity in Nasopharyngeal Carcinoma by Inhibiting Cell Cycle Arrest

Jian Zhang et al. J Exp Clin Cancer Res.

Abstract

Background: Epigenetic abnormalities play important roles in nasopharyngeal cancer (NPC), however, the epigenetic changes associated with abnormal cell proliferation remain unclear.

Methods: We detected epigenetic change of ZNF671 in NPC tissues and cell lines by bisulfite pyrosequencing. We evaluated zinc finger protein 671 (ZNF671) expression in NPC cell lines and clinical tissues using real-time PCR and western blotting. Then, we established NPC cell lines that stably overexpressed ZNF671 and knocked down ZNF671 expression to explore its function in NPC in vitro and in vivo. Additionally, we investigated the potential mechanism of ZNF671 by identifying the mitotic spindle and G2/M checkpoint pathways pathway downstream genes using gene set enrichment analysis, flow cytometry and western blotting.

Results: ZNF671 was hypermethylated in NPC tissues and cell lines. The mRNA and protein expression of ZNF671 was down-regulated in NPC tissues and cell lines and the mRNA expression could be upregulated after the demethylation agent 5-aza-2'-deoxycytidine treatment. Overexpression of ZNF671 suppressed NPC cell proliferation and colony formation in vitro; silencing ZNF671 using a siRNA had the opposite effects. Additionally, overexpression of ZNF671 reduced the tumorigenicity of NPC cells in xenograft model in vivo. The mechanism study determined that overexpressing ZNF671 induced S phase arrest in NPC cells by upregulating p21 and downregulating cyclin D1 and c-myc.

Conclusions: Epigenetic mediated zinc finger protein 671 downregulation promotes cell proliferation and enhances tumorigenicity by inhibiting cell cycle arrest in NPC, which may represent a novel potential therapeutic target.

Keywords: Hypermethylation; Nasopharyngeal carcinoma; Proliferation; Tumorigenicity; ZNF671.

Conflict of interest statement

Competing interests

The authors declare that they have no competing interests.

Ethical approval

This study was performed in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the ethics committee of Sun Yat-sen university Cancer center.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
ZNF671 is hypermethylated in NPC. a Schematic illustration of the ZNF671 promoter CpG islands and bisulfite pyrosequencing region. Input sequence: red region; CpG islands: blue region; TSS: transcription start site; cg11977686: CG site identified in our previous genome-wide methylation analysis; red text: CG sites for bisulfite pyrosequencing; bold red text: most significantly altered CG site in ZNF671. b and c Bisulfite pyrosequencing analysis of the ZNF671 promoter region (b) and the average methylation levels (c) in normal (n = 8) and NPC (n = 8) tissues. Red text: cg11977686 CG site. d Bisulfite pyrosequencing analysis of ZNF671 promoter region, as determined by bisulfite pyrosequencing analysis, in NP69 and NPC (CNE1, CNE2, SUNE1, HONE1, HNE1, 5-8F and 6-10B) cell lines. d Quantitative RT-PCR analysis of ZNF671 mRNA expression in NPC cell lines after DAC treatment. All experiments were performed at least three times; data are mean ± SD. **P < 0.01 vs. control, Student’s t-test
Fig. 2
Fig. 2
Promoter hypermethylation contributes to downregulation of ZNF671 in NPC. a Quantitative RT-PCR analysis of ZNF671 mRNA expression in NP69 and NPC cell lines. b ZNF671 mRNA is downregulated in the GSE12452 nasopharyngeal carcinoma dataset. c-d Western blotting analysis of ZNF671 in NPC (CNE1, CNE2, SUNE1, HONE1, HNE1, 5-8F and 6-10B) cell lines and NPC (T, n = 4) and normal nasopharyngeal epithelial tissues (N, n = 4). e and f ZNF671 methylation levels measured via bisulfite pyrosequencing analysis (e) and relative ZNF671 mRNA levels measured via real-time RT–PCR analysis (f) with (DAC+) or without (DAC−) DAC treatment in NP69 and NPC cell lines. All experiments were repeated at least three times; data are mean ± SD; P-values were calculated using the Student’s t-test
Fig. 3
Fig. 3
Effects of ZNF671 overexpression on NPC cell viability and colony formation ability in vitro. a qPCR analysis of ZNF671 mRNA expression in CNE-2 and 5-8F cells stably overexpression ZNF671. b Western blotting analysis of ZNF671 expression in CNE-2 and 5-8F cells stably overexpression ZNF671. c-d The CCK-8 assay showed overexpression of ZNF671 reduced the viability of CNE2 (c) and 5-8F (d) cells. e The colony formation assay showed overexpression of ZNF671 suppressed colony-forming ability. All experiments were performed at least three times; data are mean ± SD. *P < 0.05, **P < 0.01 vs. control, Student’s t-test
Fig. 4
Fig. 4
Effects of ZNF671 silencing on NPC cell viability and colony formation in vitro. a qPCR analysis of ZNF671 silence in NP69 and N2Tert cells. b Western blotting analysis of ZNF671 silence in NP69 and N2Tert cells. c-d Silencing ZNF671 increased the proliferation (c) and colony formation (d) ability of nasopharyngeal epithelial NP69 and N2Tert cells. All experiments were performed at least three times; data are mean ± SD. *P < 0.05, **P < 0.01 vs. control, Student’s t-test
Fig. 5
Fig. 5
ZNF671 reduces the tumorigenicity of NPC cells in vivo. a BALB/c mice were injected with the indicated cells. Images of the mice and tumors formed at 30 days after injection. b Growth curves of tumor volume. c Images of the excised tumors. d Excised tumor weight. Data are mean ± SD. *P < 0.05, **P < 0.01 vs. control, Student’s t-test
Fig. 6
Fig. 6
ZNF671 induces cell cycle arrest at the S phase. a GSEA enrichment plots revealed that enrichment of mitotic spindle and G2/M checkpoint pathways was associated with downregulation of ZNF671. b-c Flow cytometry analysis of cell cycle distribution in cells overexpressing ZNF671 and (c) after silencing ZNF671. d-f Western blot analysis of proteins related to the S phase in cells overexpressing ZNF671 and (e-f) after silencing ZNF671

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