Tumor Biology

, Volume 33, Issue 5, pp 1307–1317 | Cite as

Genome-wide analysis of DNA methylation identifies novel cancer-related genes in hepatocellular carcinoma

  • Masahiro Shitani
  • Shigeru Sasaki
  • Noriyuki Akutsu
  • Hideyasu Takagi
  • Hiromu Suzuki
  • Masanori Nojima
  • Hiroyuki Yamamoto
  • Takashi Tokino
  • Koichi Hirata
  • Kohzoh Imai
  • Minoru Toyota
  • Yasuhisa Shinomura
Research Article


Aberrant DNA methylation has been implicated in the development of hepatocellular carcinoma (HCC). Our aim was to clarify its molecular mechanism and to identify useful biomarkers by screening for DNA methylation in HCC. Methylated CpG island amplification coupled with CpG island microarray (MCAM) analysis was carried out to screen for methylated genes in primary HCC specimens [hepatitis B virus (HBV)-positive, n = 4; hepatitis C virus (HCV)-positive, n = 5; HBV/HCV-negative, n = 7]. Bisulfite pyrosequencing was used to analyze the methylation of selected genes and long interspersed nuclear element (LINE)-1 in HCC tissue (n = 57) and noncancerous liver tissue (n = 50) from HCC patients and in HCC cell lines (n = 10). MCAM analysis identified 332, 342, and 259 genes that were methylated in HBV-positive, HCV-positive, and HBV/HCV-negative HCC tissues, respectively. Among these genes, methylation of KLHL35, PAX5, PENK, and SPDYA was significantly higher in HCC tissue than in noncancerous liver tissue, irrespective of the hepatitis virus status. LINE-1 hypomethylation was also prevalent in HCC and correlated positively with KLHL35 and SPDYA methylation. Receiver operating characteristic curve analysis revealed that methylation of the four genes and LINE-1 strongly discriminated between HCC tissue and noncancerous liver tissue. Our data suggest that aberrant hyper- and hypomethylation may contribute to a common pathogenesis mechanism in HCC. Hypermethylation of KLHL35, PAX, PENK, and SDPYA and hypomethylation of LINE-1 could be useful biomarkers for the detection of HCC.


Hepatocellular carcinoma DNA methylation CpG island LINE-1 Biomarker 



We thank Dr. Yutaka Kondo for technical advice on MCAM analysis and Masami Ashida for technical assistance. This study was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for Promotion of Science (Y. Shinomura), a Grant-in-Aid for the Third-term Comprehensive 10-year Strategy for Cancer Control (M. Toyota and H. Suzuki), and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare, Japan (M. Toyota and H. Suzuki).

Conflicts of interest


Supplementary material

13277_2012_378_MOESM1_ESM.xls (36 kb)
Supplementary Table 1 Primer sequences used in this study (XLS 36 kb)
13277_2012_378_MOESM2_ESM.xls (29 kb)
Supplementary Table 2 GO analysis of commonly methylated genes in HCC (XLS 29 kb)
13277_2012_378_MOESM3_ESM.pdf (936 kb)
ESM 1 (PDF 936 kb)


  1. 1.
    Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74–108.CrossRefPubMedGoogle Scholar
  2. 2.
    Chen CJ, Yu MW, Liaw YF. Epidemiological characteristics and risk factors of hepatocellular carcinoma. J Gastroenterol Hepatol. 1997;12:S294–308.CrossRefPubMedGoogle Scholar
  3. 3.
    Montesano R, Hainaut P, Wild CP. Hepatocellular carcinoma: from gene to public health. J Natl Cancer Inst. 1997;89:1844–51.CrossRefPubMedGoogle Scholar
  4. 4.
    Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet. 2002;31:339–46.CrossRefPubMedGoogle Scholar
  5. 5.
    Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002;3:415–28.CrossRefPubMedGoogle Scholar
  6. 6.
    Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10:691–703.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 2004;32:e38.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Takai D, Yagi Y, Habib N, Sugimura T, Ushijima T. Hypomethylation of line1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis. Jpn J Clin Oncol. 2000;30:306–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Kim MJ, White-Cross JA, Shen L, Issa JP, Rashid A. Hypomethylation of long interspersed nuclear element-1 in hepatocellular carcinomas. Mod Pathol. 2009;22:442–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Kaneto H, Sasaki S, Yamamoto H, Itoh F, Toyota M, Suzuki H, Ozeki I, Iwata N, Ohmura T, Satoh T, Karino Y, Toyota J, Satoh M, Endo T, Omata M, Imai K. Detection of hypermethylation of the p16(ink4a) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut. 2001;48:372–7.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Takagi H, Sasaki S, Suzuki H, Toyota M, Maruyama R, Nojima M, Yamamoto H, Omata M, Tokino T, Imai K, Shinomura Y. Frequent epigenetic inactivation of sfrp genes in hepatocellular carcinoma. J Gastroenterol. 2008;43:378–89.CrossRefPubMedGoogle Scholar
  12. 12.
    Lee HS, Kim BH, Cho NY, Yoo EJ, Choi M, Shin SH, Jang JJ, Suh KS, Kim YS, Kang GH. Prognostic implications of and relationship between CpG island hypermethylation and repetitive DNA hypomethylation in hepatocellular carcinoma. Clin Cancer Res. 2009;15:812–20.CrossRefPubMedGoogle Scholar
  13. 13.
    Gao W, Kondo Y, Shen L, Shimizu Y, Sano T, Yamao K, Natsume A, Goto Y, Ito M, Murakami H, Osada H, Zhang J, Issa JP, Sekido Y. Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas. Carcinogenesis. 2008;29:1901–10.CrossRefPubMedGoogle Scholar
  14. 14.
    Arai E, Ushijima S, Gotoh M, Ojima H, Kosuge T, Hosoda F, Shibata T, Kondo T, Yokoi S, Imoto I, Inazawa J, Hirohashi S, Kanai Y. Genome-wide DNA methylation profiles in liver tissue at the precancerous stage and in hepatocellular carcinoma. Int J Cancer. 2009;125:2854–62.CrossRefPubMedGoogle Scholar
  15. 15.
    Deng YB, Nagae G, Midorikawa Y, Yagi K, Tsutsumi S, Yamamoto S, Hasegawa K, Kokudo N, Aburatani H, Kaneda A. Identification of genes preferentially methylated in hepatitis C virus-related hepatocellular carcinoma. Cancer Sci. 2010;101:1501–10.CrossRefPubMedGoogle Scholar
  16. 16.
    Goto Y, Shinjo K, Kondo Y, Shen L, Toyota M, Suzuki H, Gao W, An B, Fujii M, Murakami H, Osada H, Taniguchi T, Usami N, Kondo M, Hasegawa Y, Shimokata K, Matsuo K, Hida T, Fujimoto N, Kishimoto T, Issa JP, Sekido Y. Epigenetic profiles distinguish malignant pleural mesothelioma from lung adenocarcinoma. Cancer Res. 2009;69:9073–82.CrossRefPubMedGoogle Scholar
  17. 17.
    Suzuki H, Yamamoto E, Nojima M, Kai M, Yamano HO, Yoshikawa K, Kimura T, Kudo T, Harada E, Sugai T, Takamaru H, Niinuma T, Maruyama R, Yamamoto H, Tokino T, Imai K, Toyota M, Shinomura Y. Methylation-associated silencing of microrna-34b/c in gastric cancer and its involvement in an epigenetic field defect. Carcinogenesis. 2010;31:2066–73.CrossRefPubMedGoogle Scholar
  18. 18.
    Nishida N, Nagasaka T, Nishimura T, Ikai I, Boland CR, Goel A. Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology. 2008;47:908–18.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wong IH, Zhang J, Lai PB, Lau WY, Lo YM. Quantitative analysis of tumor-derived methylated p16ink4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients. Clin Cancer Res. 2003;9:1047–52.PubMedGoogle Scholar
  20. 20.
    Zhang YJ, Wu HC, Shen J, Ahsan H, Tsai WY, Yang HI, Wang LY, Chen SY, Chen CJ, Santella RM. Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA. Clin Cancer Res. 2007;13:2378–84.CrossRefPubMedGoogle Scholar
  21. 21.
    Carotta S, Holmes ML, Pridans C, Nutt SL. Pax5 maintains cellular identity by repressing gene expression throughout B cell differentiation. Cell Cycle. 2006;5:2452–6.CrossRefPubMedGoogle Scholar
  22. 22.
    Palmisano WA, Crume KP, Grimes MJ, Winters SA, Toyota M, Esteller M, Joste N, Baylin SB, Belinsky SA. Aberrant promoter methylation of the transcription factor genes pax5 alpha and beta in human cancers. Cancer Res. 2003;63:4620–5.PubMedGoogle Scholar
  23. 23.
    Lazzi S, Bellan C, Onnis A, De Falco G, Sayed S, Kostopoulos I, Onorati M, D’Amuri A, Santopietro R, Vindigni C, Fabbri A, Righi S, Pileri S, Tosi P, Leoncini L. Rare lymphoid neoplasms coexpressing B- and T-cell antigens. The role of pax-5 gene methylation in their pathogenesis. Hum Pathol. 2009;40:1252–61.CrossRefPubMedGoogle Scholar
  24. 24.
    Liu W, Li X, Chu ES, Go MY, Xu L, Zhao G, Li L, Dai N, Si J, Tao Q, Sung JJ, Yu J. Paired box gene 5 is a novel tumor suppressor in hepatocellular carcinoma through interaction with p53 signaling pathway. Hepatology. 2011;53:843–53.CrossRefPubMedGoogle Scholar
  25. 25.
    Li X, Cheung KF, Ma X, Tian L, Zhao J, Go MY, Shen B, Cheng AS, Ying J, Tao Q, Sung JJ, Kung HF, Yu J. Epigenetic inactivation of paired box gene 5, a novel tumor suppressor gene, through direct upregulation of p53 is associated with prognosis in gastric cancer patients. Oncogene. 2011. doi: 10.1038/onc.2011.511.
  26. 26.
    Morris MR, Ricketts CJ, Gentle D, McRonald F, Carli N, Khalili H, Brown M, Kishida T, Yao M, Banks RE, Clarke N, Latif F, Maher ER. Genome-wide methylation analysis identifies epigenetically inactivated candidate tumour suppressor genes in renal cell carcinoma. Oncogene. 2011;30:1390–401.CrossRefPubMedGoogle Scholar
  27. 27.
    Ueki T, Toyota M, Skinner H, Walter KM, Yeo CJ, Issa JP, Hruban RH, Goggins M. Identification and characterization of differentially methylated cpg islands in pancreatic carcinoma. Cancer Res. 2001;61:8540–6.PubMedGoogle Scholar
  28. 28.
    Goo YA, Goodlett DR, Pascal LE, Worthington KD, Vessella RL, True LD, Liu AY. Stromal mesenchyme cell genes of the human prostate and bladder. BMC Urol. 2005;5:17.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Chung JH, Lee HJ, Kim BH, Cho NY, Kang GH. DNA methylation profile during multistage progression of pulmonary adenocarcinomas. Virchows Arch. 2011;459:201–11.CrossRefPubMedGoogle Scholar
  30. 30.
    Chung W, Bondaruk J, Jelinek J, Lotan Y, Liang S, Czerniak B, Issa JP. Detection of bladder cancer using novel DNA methylation biomarkers in urine sediments. Cancer Epidemiol Biomarkers Prev. 2011;20:1483–91.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kishida Y, Natsume A, Kondo Y, Takeuchi I, An B, Okamoto Y, Shinjo K, Saito K, Ando H, Ohka F, Sekido Y, Wakabayashi T. Epigenetic subclassification of meningiomas based on genome-wide DNA methylation analyses. Carcinogenesis. 2012;33:436–41.Google Scholar
  32. 32.
    McTavish N, Copeland LA, Saville MK, Perkins ND, Spruce BA. Proenkephalin assists stress-activated apoptosis through transcriptional repression of nf-kappab- and p53-regulated gene targets. Cell Death Differ. 2007;14:1700–10.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Gastwirt RF, McAndrew CW, Donoghue DJ. Speedy/ringo regulation of CDKs in cell cycle, checkpoint activation and apoptosis. Cell Cycle. 2007;6:1188–93.CrossRefPubMedGoogle Scholar
  34. 34.
    Zucchi I, Mento E, Kuznetsov VA, Scotti M, Valsecchi V, Simionati B, Vicinanza E, Valle G, Pilotti S, Reinbold R, Vezzoni P, Albertini A, Dulbecco R. Gene expression profiles of epithelial cells microscopically isolated from a breast-invasive ductal carcinoma and a nodal metastasis. Proc Natl Acad Sci U S A. 2004;101:18147–52.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Golipour A, Myers D, Seagroves T, Murphy D, Evan GI, Donoghue DJ, Moorehead RA, Porter LA. The spy1/ringo family represents a novel mechanism regulating mammary growth and tumorigenesis. Cancer Res. 2008;68:3591–600.CrossRefPubMedGoogle Scholar
  36. 36.
    Ke Q, Ji J, Cheng C, Zhang Y, Lu M, Wang Y, Zhang L, Li P, Cui X, Chen L, He S, Shen A. Expression and prognostic role of spy1 as a novel cell cycle protein in hepatocellular carcinoma. Exp Mol Pathol. 2009;87:167–72.CrossRefPubMedGoogle Scholar
  37. 37.
    Lin CH, Hsieh SY, Sheen IS, Lee WC, Chen TC, Shyu WC, Liaw YF. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res. 2001;61:4238–43.PubMedGoogle Scholar
  38. 38.
    Park IY, Sohn BH, Yu E, Suh DJ, Chung YH, Lee JH, Surzycki SJ, Lee YI. Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology. 2007;132:1476–94.CrossRefPubMedGoogle Scholar
  39. 39.
    Calvisi DF, Simile MM, Ladu S, Pellegrino R, De Murtas V, Pinna F, Tomasi ML, Frau M, Virdis P, De Miglio MR, Muroni MR, Pascale RM, Feo F. Altered methionine metabolism and global DNA methylation in liver cancer: relationship with genomic instability and prognosis. Int J Cancer. 2007;121:2410–20.CrossRefPubMedGoogle Scholar
  40. 40.
    Tangkijvanich P, Hourpai N, Rattanatanyong P, Wisedopas N, Mahachai V, Mutirangura A. Serum line-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma. Clin Chim Acta. 2007;379:127–33.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2012

Authors and Affiliations

  • Masahiro Shitani
    • 1
  • Shigeru Sasaki
    • 1
  • Noriyuki Akutsu
    • 1
  • Hideyasu Takagi
    • 1
  • Hiromu Suzuki
    • 1
    • 2
  • Masanori Nojima
    • 3
  • Hiroyuki Yamamoto
    • 1
  • Takashi Tokino
    • 4
  • Koichi Hirata
    • 5
  • Kohzoh Imai
    • 6
  • Minoru Toyota
    • 2
  • Yasuhisa Shinomura
    • 1
  1. 1.First Department of Internal MedicineSapporo Medical UniversitySapporoJapan
  2. 2.Department of Molecular BiologySapporo Medical UniversitySapporoJapan
  3. 3.Department of Public HealthSapporo Medical UniversitySapporoJapan
  4. 4.Medical Genome Science, Research Institute for Frontier MedicineSapporo Medical University School of MedicineSapporoJapan
  5. 5.First Department of SurgerySapporo Medical UniversitySapporoJapan
  6. 6.Division of Novel Therapy for Cancer, The Advanced Clinical Research Center, The Institute of Medical ScienceThe University of TokyoTokyoJapan

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