Ann Hepatobiliary Pancreat Surg.  2017 Nov;21(4):181-187. 10.14701/ahbps.2017.21.4.181.

Promoter methylation of cysteine dioxygenase type 1: gene silencing and tumorigenesis in hepatocellular carcinoma

• Choi JI
• Cho EH
• Kim SB
• Kim R
• Kwon J
• Park M
• Shin HJ
• Ryu HS
• Park SH
• Lee KH

• KMID: 2397796
• DOI: http://doi.org/10.14701/ahbps.2017.21.4.181

Abstract

BACKGROUNDS/AIMS
Cysteine dioxygenase type 1 (CDO1) acts as a tumor suppressor and is silenced by promoter methylation in various malignancies. The relationship between the CDO1 methylation status and hepatocellular carcinoma (HCC) tumorigenesis was evaluated.
METHODS
Using a HCC cell line (SNU423), an in vitro demethylation study was performed to confirm whether promoter methylation causes CDO1 down-regulation. The SNU423 cells transfected with the CDO1 cell function was compared to that of naïve cells. An in vivo study using immunohistochemical staining of HCC specimens that were collected from patients who underwent curative liver resection was also performed.
RESULTS
CDO1 was activated after demethylation treatment in the HCC specimens. Moreover, tumor cell proliferation, colony-forming, migration, and invasion activities significantly decreased after CDO1 transfection (p < 0.05). The percentage of tumors that were larger than 5 cm was higher in patients who had a lower expression of CDO1 (p=0.030). Vascular invasion and histological grade were independent prognostic factors for poor overall and recurrence-free survival. The degree of CDO1 expression was not an independent prognostic factor in this study's population.
CONCLUSIONS
These results suggested that methylation down-regulated CDO1 expression in the HCC cells. CDO1 methylation may be a potentially valuable diagnostic biomarker for HCC.

Keyword

Hepatocellular carcinoma; Cysteine dioxygenase type 1; DNA methylation; Demethylation; Biomarker

MeSH Terms

Carcinogenesis*
Carcinoma, Hepatocellular*
Cell Line
Cell Proliferation
Cysteine Dioxygenase*
Cysteine*
DNA Methylation
Down-Regulation
Gene Silencing*
Humans
In Vitro Techniques
Liver
Methylation*
Transfection
Cysteine
Cysteine Dioxygenase

Figure

• 

  Fig. 1 CDO1 expression. CDO1 expression was evaluated in the HCC cell lines with and without demethylation (5-Aza-dC) treatment by RT-PCR. (GAPDH, glyceraldehyde 3-phospate dehydrogenase: housekeeping gene).

• 

  Fig. 2 Analyses of CDO1 protein in the SNU423 HCC cell line with and without pCDNA3-CDO1 transfection (CDO1 overexpression). (A) Western blot analysis of CDO1 protein in SNU423 HCC cells; (B) Cell proliferation assay: The proliferation activities of HCC cells with pCDNA3-CDO1 transfection were suppressed at day 5 (p<0.025) and at day 7 (p<0.003); (C) Colony-forming assay: The colony-forming activities of the HCC cells with pCDNA3-CDO1 transfection were suppressed after 2 weeks (p<0.014); (D) migration assay; (E) invasion assay.

• 

  Fig. 3 Immunohistochemical analysis for the detection of the CDO1 protein in HCC tissues. Representative samples of the different staining intensities are displayed.

• 

  Fig. 4 Dot plot based on the immunostaining intensity and proportion of the stained cells stained at each score.

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