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Identification of GPX3 Epigenetically Silenced by CpG Methylation in Human Esophageal Squamous Cell Carcinoma

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Abstract

Background

Esophageal squamous cell carcinoma (ESCC) is one of the most common causes of cancer mortality in the gastrointestinal tract. Promoter hypermethylation of tumor suppressor genes contributes to gene inactivation during development of ESCC.

Aim

To identify novel methylation-silenced genes in ESCC.

Methods

Genome-wide microarrays were applied to search for genes that were markedly upregulated after treatment with 5-aza-2′-deoxycytidine (5-Aza-dC) and that were markedly decreased in tumor tissue compared with paired adjacent nontumor tissue. Reverse-transcription polymerase chain reaction (PCR), immunohistochemistry, methylation-specific PCR, and bisulfite genomic sequencing were employed to investigate expression and methylation of candidate genes in five human ESCC cell lines, two human immortalized normal esophageal epithelial cell lines, primary ESCC tumor tissues, and paired adjacent nontumor tissues.

Results

GPX3 was selected as a novel candidate hypermethylated gene in ESCC through microarray analysis. In most ESCC cell lines, GPX3 messenger RNA (mRNA) expression was downregulated and the CpG island of GPX3 promoter was methylated. Demethylation treatment with 5-Aza-dC restored GPX3 mRNA expression. Methylation of GPX3 promoter was more frequent in ESCC tumor tissues (71.4%) than in adjacent nontumor tissues (10.7%) (P < 0.001), and methylation of GPX3 promoter correlated significantly with GPX3 mRNA downregulation. Finally, GPX3 protein expression was also significantly lower in ESCC tumor tissues than in adjacent nontumor tissues.

Conclusion

GPX3 is downregulated through promoter hypermethylation in ESCC, which may be a potential biomarker of ESCC.

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Abbreviations

ESCC:

Esophageal squamous cell carcinoma

GPX3:

Glutathione peroxidase 3

UCHL1:

Ubiquitin carboxyl-terminal esterase L1

TSPYL5:

TSPY-like 5

PPP1R14A:

Protein phosphatase 1 regulatory (inhibitor) subunit 14A

DLC1:

Deleted in liver cancer 1

5-Aza-dC:

5-Aza-2′-deoxycytidine

ECRG4:

Esophageal cancer related gene 4 protein

APC:

Adenomatous polyposis coli

HPP1:

Hyperplastic polyposis protein 1

RT-PCR:

Reverse-transcription PCR

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

MSP:

Methylation-specific PCR

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Acknowledgments

This work was supported by National ‘863’ High-tech Program Grant in China (2007AA02Z4Z4), China Postdoctoral Science Foundation (20090460394), and Beijing Municipal Natural Science Foundation (7072022).

Conflict of interest

There is no conflict of interest.

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Authors and Affiliations

  1. Department of Gastroenterology, Beijing Friendship Hospital Affiliated to the Capital Medical University, Beijing Digestive Disease Center, 95 Yong’an Road, 100050, Beijing, People’s Republic of China

    Yuanlong He, Yongjun Wang, Peng Li, Shengtao Zhu, Junxiong Wang & Shutian Zhang

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  1. Yuanlong He
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  2. Yongjun Wang
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  3. Peng Li
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  4. Shengtao Zhu
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  5. Junxiong Wang
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  6. Shutian Zhang
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Corresponding author

Correspondence to Shutian Zhang.

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He, Y., Wang, Y., Li, P. et al. Identification of GPX3 Epigenetically Silenced by CpG Methylation in Human Esophageal Squamous Cell Carcinoma. Dig Dis Sci 56, 681–688 (2011). https://doi.org/10.1007/s10620-010-1369-0

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  • DOI: https://doi.org/10.1007/s10620-010-1369-0

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