Identification of genes and pathways, including the CXCL2 axis, altered by DNA methylation in hepatocellular carcinoma

  • Sophia Subat
  • Kaoru Mogushi
  • Mahmut Yasen
  • Takashi Kohda
  • Yuichi IshikawaEmail author
  • Hiroshi Tanaka
Original Article – Cancer Research



Recent genetic studies have suggested that tumor suppressor genes are often silenced during carcinogenesis via epigenetic modification caused by methylation of promoter CpG islands. Here, we characterized genes inactivated by DNA methylation in human hepatocellular carcinoma (HCC) to identify the genes and pathways involved in DNA methylation in hepatocellular carcinoma.


Eight HCC-derived cell lines were treated with a DNA demethylating agent, 5-aza-2′-deoxycytidine. Additionally, 100 pairs of primary HCC and adjacent non-cancerous tissues as well as 15 normal liver tissues were analyzed by comprehensive gene expression analysis using microarrays. Moreover, gene set enrichment analysis identified the major molecular pathways associated with DNA methylation. Validation of gene expression and DNA methylation status was performed by real-time PCR after bisulfite modification.


We showed that CXCL2, an immune-related chemokine, expression was significantly downregulated in tumor tissues and also significantly upregulated by DAC treatment in cell lines. Furthermore, we observed a statistically significant difference in methylation status between normal liver tissues and tumor tissues (P < 0.05). In addition, tumors with higher CXCL2 expression included significantly more numbers of multiple tumors than the lower expression group.


We identified CXCL2, an immune-related chemokine, decreased in hepatocellular carcinoma and the regulation mechanism may be controlled by methylation. Further studies should be warranted to examine if and to what extent the gene is actually suppressed by methylation and if there is a possibility that the CXCL2 axis plays a role for diagnosis and treatment of hepatocellular carcinoma.


Hepatocellular carcinoma DNA methylation Tumor suppressor genes CXCL2 Inflammation 



Activating transcription factor 3


CpG islands


Chemokine (C-X-C motif) ligand 2




Epithelial–mesenchymal transition


Fold change


False discovery rate


Growth-related oncogene


Gene set enrichment analysis


Hepatocellular carcinoma


Jun B proto-oncogene


Kyoto Encyclopedia of Genes and Genomes


Methylation specific PCR


Overall survival


Polymerase chain reaction


Prostaglandin-endoperoxide synthase 2


Recurrence-free survival


Robust multiarray average


Standard deviation


Transcription factor binding site


Transforming growth factor-β


Thrombospondin 1


Tumor suppressor genes


Authors’ contributions

All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization: SS, MY and HT Methodology, KM, MY and TK Investigation, SS, KM and MY Formal Analysis, SS and KM Resources, KM, TK and HT Writing—Original Draft, SS Writing-Review & Editing, KM and YI Visualization, SS Supervision, KM and YI Funding Acquisition KM and YI.


This work was supported partly by JSPS KAKENHI Grant Number: JP26870175 (KM), JSPS KAKENHI Grant Number: 15H04714 (YI).

Compliance with ethical standards

Conflict of interest

Yuichi Ishikawa received research grants from Daiichi Sankyo Co., Ltd. and Ono Pharmaceutical Co., Ltd., as well as he is a consultant of Fujirebio Inc. All other authors declare no conflicts of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

432_2018_2824_MOESM1_ESM.docx (617 kb)
Supplementary material 1 (DOCX 617 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Systems Biology, Graduate School of Biomedical ScienceTokyo Medical and Dental UniversityTokyoJapan
  2. 2.Department of Epigenetics, Graduate School of Biomedical ScienceTokyo Medical and Dental UniversityTokyoJapan
  3. 3.Division of PathologyThe Cancer Institute, Japanese Foundation for Cancer ResearchTokyoJapan
  4. 4.Intractable Disease Research Center, Graduate School of MedicineJuntendo UniversityTokyoJapan
  5. 5.Department of SurgeryXinjiang Medical University Affiliated Tumor HospitalUrumqiChina

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