Association of hypomethylation of LINE-1 repetitive element in blood leukocyte DNA with an increased risk of hepatocellular carcinoma

Association of hypomethylation of LINE-1 repetitive element in blood leukocyte DNA with an increased risk of hepatocellular carcinoma 1 first-author 1 2 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhangpindjz@163.com †E-mail:zhangpindjz@163.com zhangpindjz@163.com 3 4 Department of General Surgery, the Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China Department of General Surgery, the Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China Department of Gastroenterology, Shanghai Guanghua Hospital, Shanghai 200052, China Department of Gastroenterology, Shanghai Guanghua Hospital, Shanghai 200052, China Department of General Surgery, Tongji Hospital, Tongji University, Shanghai 200065, China Department of General Surgery, Tongji Hospital, Tongji University, Shanghai 200065, China Department of Oncology, No. 113 Hospital of People’s Liberation Army, Ningbo 315040, China Department of Oncology, No. 113 Hospital of People’s Liberation Army, Ningbo 315040, China Global DNA hypomethylation has been associated with increased risk for cancers of the colorectum, bladder, breast, head and neck, and testicular germ cells. The aim of this study was to examine whether global hypomethylation in blood leukocyte DNA is associated with the risk of hepatocellular carcinoma (HCC). A total of 315 HCC cases and 356 age-, sex- and HBsAg status-matched controls were included. Global methylation in blood leukocyte DNA was estimated by analyzing long interspersed element-1 (LINE-1) repeats using bisulfite-polymerase chain reaction (PCR) and pyrosequencing. We observed that the median methylation level in HCC cases (percentage of 5-methylcytosine (5mC)=77.7%) was significantly lower than that in controls (79.5% 5mC) ( P =0.004, Wilcoxon rank-sum test). The odds ratios (ORs) of HCC for individuals in the third, second, and first (lowest) quartiles of LINE-1 methylation were 1.1 (95% confidence interval (CI) 0.7–1.8), 1.4 (95% CI 0.8–2.2), and 2.6 (95% CI 1.7–4.1) ( P for trend < 0.001), respectively, compared to individuals in the fourth (highest) quartile. A 1.9-fold (95% CI 1.4–2.6) increased risk of HCC was observed among individuals with LINE-1 methylation below the median compared to individuals with higher ( > median) LINE-1 methylation. Our results demonstrate for the first time that individuals with global hypomethylation measured in LINE-1 repeats in blood leukocyte DNA have an increased risk for HCC. Our data provide the evidence that global hypomethylation detected in the easily obtainable DNA source of blood leukocytes may help identify individuals at risk of HCC. Cancer risk Epigenetics Global hypomethylation Hepatocellular carcinoma LINE-1 repetitive element Introduction 1 Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, while ranks the third most common cause of cancer-related death (Parkin et al., 2005 2005 ; Gomaa et al., 2008 2008 ). Its overall incidence remains high especially in the developing world and is steadily rising across most of the developed world (Shariff et al., 2009 2009 ). Chronic infection with hepatitis B or C virus (HBV or HCV), aflatoxin exposure, liver cirrhosis, chronical alcohol consumption, and genetic factors have been characterized to play a major role in the HCC etiology (Morgan et al., 2004 2004 ; Kim and Lee, 2005 2005 ; Parkin et al., 2005 2005 ; Okuda, 2007 2007 ; Gomaa et al., 2008 2008 ; Shariff et al., 2009 2009 ). Epigenetic changes in methylation patterns are increasingly implicated in cancer development (Feinberg and Tycko, 2004 2004 ). Global DNA hypomethylation seems to occur in early neoplasia and has been regarded as an important component of cancer development (Das and Singal, 2004 2004 ; Zhu et al., 2011 2011 ). Compared with normal tissue counterparts, lower global DNA methylation was observed in tumor tissues in a broad panel of cancers including HCC (Das and Singal, 2004 2004 ; Formeister et al., 2010 2010 ). Animal studies have shown that experimentally-induced hypomethylation can lead to cancer development at multiple sites (Wilson et al., 1984 1984 ; Thomas and Williams, 1992 1992 ; Gaudet et al., 2003 2003 ), indicating the causal role of global DNA hypomethylation in cancer development. In addition, lower levels of global methylation were reported in leukocyte DNA from subjects with colorectal adenoma, the precursor of colorectal cancer (Pufulete et al., 2003 2003 ; Lim et al., 2008 2008 ). Further, in healthy populations, global hypomethylation in blood leukocyte DNA has been associated with exposure to environmental risk factors for carcinogenesis, such as benzene, tobacco smoke, persistent organic pollutants, and perfluorooctane sulfonate (Bollati et al., 2007 2007 ; Rusiecki et al., 2008 2008 ; Breton et al., 2009 2009 ; Liu et al., 2010 2010 ; Wan et al., 2010 2010 ). Several case-control studies have shown that global hypomethylation measured in blood DNA was associated with an increased risk for cancers of the colorectum, bladder, breast, head and neck, and testicular germ cells (Pufulete et al., 2003 2003 ; Hsiung et al., 2007 2007 ; Moore et al., 2008 2008 ; Choi et al., 2009 2009 ; Cho et al., 2010 2010 ; Mirabello et al., 2010 2010 ), suggesting that global DNA hypomethylation is a potential biomarker of cancer susceptibility. Global DNA methylation level derives from the overall 5-methylcytosine (5mC) dinucleotide CpG sites in the human genome, about 55% of which consists of repetitive elements (Lander et al., 2001 2001 ), including approximately 500 000 long interspersed element-1 (LINE-1) repeats which represent appro-ximately 17% of the human genome (Cordaux and Batzer, 2009 2009 ). Because of heavy methylation in normal tissue, high representation throughout the genome, and close correlation with genomic DNA methylation content, LINE-1 methylation status has been used as a surrogate marker for estimating the genomic DNA methylation level (Yang et al., 2004 2004 ; Weisenberger et al., 2005 2005 ). In the present case-control study based on 315 HCC cases and 356 controls from a Chinese population, we sought to determine the association between LINE-1 hypomethylation and the risk of HCC. Materials and methods 1 Subjects 2 This is a hospital-based case-control study. A total of 315 newly diagnosed HCC patients were recruited from the Sixth People’s Hospital of Shanghai Jiao Tong University and Tongji Hospital of Tongji University, Shanghai, China from February 2003 to October 2009. All cases did not receive chemotherapy or radiation therapy before blood sample collection. Final diagnoses were pathologically confirmed from the specimens obtained by surgery. As a control, blood samples of 356 cancer-free subjects were randomly selected from the individuals who attended for the physical examination at the same hospitals during the period when the case patients were recruited. The controls were frequency-matched to the cases by age (±5 years), sex, and serum HBsAg status. For both case and control subjects, those with other cancers, diabetes, autoimmune disorders, or other major systemic diseases were excluded. Information on age, sex, habits of alcohol drinking and cigarette smoking, and HCC family history was obtained using structured questionnaire through in-person interviews. An ever-drinker was defined as a person who reported drinking alcoholic beverages at least once per week for at least six months. An ever-smoker was defined as a smoker of at least one cigarette per day for at least six months. Chronic infections of HBV and HCV were considered if the seropositivities of HBsAg and anti-HCV were detected, respectively. Tumor characteristics including tumor size, Edmondson-Steiner grade, and tumor stage (tumour-node-metastasis (TNM) staging, 6th Ed. (Lei et al., 2006 2006 )) were made on the basis of medical records. Written informed consent was obtained from each participant. The study protocol was approved by the ethics review committee of the Institutional Review Board of the two participant hospitals. DNA extraction and bisulfite modification 2 Genomic DNA of blood leukocytes was extracted using the QIAamp DNA Blood Mini kit (Qiagen, Shanghai, China). Extracted DNA was treated with sodium bisulfite using the EZ DNA Methylation kit (Zymo, CA, USA) according to the manufacturer’s protocol. LINE-1 polymerase chain reaction (PCR) and pyrosequencing 2 A modified method of PCR pyrosequencing originally described by Yang et al. ( 2004 2004 ) was performed to quantitate methylation of the repetitive LINE-1 sequences. In brief, PCR was carried out in a 50-μl reaction volume containing 25 μl of GoTaq Green Master mix (Promega, WI, USA), 1 pmol of forward primer (TTT TGA GTT AGG TGT GGG ATA TA), 1 pmol of biotinylated reverse primer (biotin-AAA ATC AAA AAA TTC CCT TTC), and 50 ng of bisulphite-treated DNA. PCR conditions were 40 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s. PCR product was bound to streptavidin sepharose beads (Amersham Biosciences, Uppsala, Sweden), and then was purified, washed, denatured, and washed again. Then, 0.3 μmol/L pyrosequencing primer (AGT TAG GTG TGG GAT ATA GT) was annealed to the purified PCR product. Pyrosequencing actions were performed in the PSQ HS 96 Pyrosequencing System. The non-CpG cytosine residues in LINE-1 repetitive sequences, which had been documented to be rarely methylated (Burden et al., 2005 2005 ), were used as built-in controls to verify bisulfite conversion, and complete conversion of cytosine at non-CpG sites ensured successful bisulfite conversion. The percentage of 5mC at each of three CpG dinucleotide positions in LINE-1 repetitive sequences, as described in detail by Tarantini et al. ( 2009 2009 ), was measured. The degree of LINE-1 methylation was expressed as percentage of methylated cytosines divided by the sum of methylated and unmethylated cytosines (percentage of 5mC). To validate PCR-pyrosequencing assay, each CpG dinucleotide position was assayed in duplicate and their averages were used in final analysis. The within-sample coefficient of variation was 0.68%, which suggests good reliability in the measurement. Statistical analysis 2 Differences between HCC cases and controls in age, sex, alcohol drinking, cigarette smoking, HBsAg status, anti-HCV status, and HCC family history were evaluated using the χ 2 -test. The nonparametric comparisons of median LINE-1 methylation levels between HCC cases and controls were evaluated by Wilcoxon rank-sum test. The association of LINE-1 methylation with HCC risk was estimated using the odds ratios (ORs) and 95% confidence intervals (CIs) from multivariate logistic regression analyses, with adjustment for age, sex, alcohol drinking, cigarette smoking, HBsAg status, anti-HCV status, and HCC family history. Statistical analyses were conducted using the Stata 10.1 (Stata Corp., College Station, TX). All tests were two-sided and a P value of ≤0.05 was considered statistically significant. Results 1 The general characteristics of the HCC cases and controls are shown in Table 1 1 . No significant differences between HCC cases and controls were found in the distribution of age, sex, alcohol drinking, cigarette smoking, or HBsAg status. Compared with controls, more cases were anti-HCV positive ( P =0.01) and had HCC family history ( P =0.04). Data are expressed as n (%) χ 2 -test Family history of HCC in first-degree relatives 2 Variable 2 Number of subjects a a 2 P b b HCC cases ( n =315) Controls ( n =356) Age  ≤50 years 143 (45.4%) 167 (46.9%) 2 0.70  >50 years 172 (54.6%) 189 (53.1%) Sex  Female 52 (16.5%) 59 (16.6%) 2 0.98  Male 263 (83.5%) 297 (83.4%) Alcohol drinking  Never 195 (61.9%) 218 (61.2%) 2 0.86  Ever 120 (38.1%) 138 (38.8%) Cigarette smoking  Never 171 (54.3%) 206 (57.9%) 2 0.35  Ever 144 (45.7%) 150 (42.1%) HBsAg  Negative 42 (13.3%) 48 (13.5%) 2 0.96  Positive 273 (86.7%) 308 (86.5%) Anti-HCV  Negative 298 (94.6%) 350 (98.3%) 2 0.01  Positive 17 (5.4%) 6 (1.7%) HCC family history c c  No 295 (93.7%) 345 (96.9%) 2 0.04  Yes 20 (6.3%) 11 (3.1%) Tumor size  ≤5 cm 155 (49.2%)  >5 cm 160 (50.8%) 2 Edmondson-Steiner grade  I/II 64 (20.3%)  III/IV 251 (79.7%) Tumor stage  I/II 227 (72.1%)  III/IV 88 (27.9%) Fig. 1 1 depicts the distribution of the LINE-1 methylation levels in HCC cases and controls as box plots of the data. The median methylation level (77.7% 5mC) in cases was significantly lower than that (79.5% 5mC) in controls ( P =0.004). Logistic regression analysis showed that the levels of LINE-1 methylation were inversely associated with the risk of HCC (Table 2 2 ). The ORs of HCC for individuals in the third, second and first (lowest) quartiles of LINE-1 methylation were 1.1 (95% CI 0.7–1.8), 1.4 (95% CI 0.8–2.2) and 2.6 (95% CI 1.7–4.1) ( P for trend < 0.001), respectively, compared to individuals in the fourth (highest) quartile. A 1.9-fold (95% CI 1.4–2.6) increased risk of HCC was observed among individuals with LINE-1 methylation below the median, compared to individuals with higher ( > median) LINE-1 methylation. Data are expressed as n (%) Adjusted for age, sex, alcohol drinking, cigarette smoking, HBsAg status, anti-HCV status, and HCC family history The quartiles and the median of LINE-1 measures were based on values among control subjects Reference P for trend < 0.001 for the ORs of HCC from the fourth, third, second to first quartiles of LINE-1 methylation 2 LINE-1 (% 5mC) 2 Number of subjects a a 2 OR (95% CI) b b 2 P b b HCC cases Controls Quartile c c Q4 52 (16.5%) 88 (24.7%) 1.0 d d Q3 58 (18.4%) 89 (25.0%) 1.1 (0.7–1.8) 0.68 Q2 70 (22.2%) 90 (25.3%) 1.4 (0.8–2.2) 0.22 Q1 135 (42.9%) 89 (25.0%) 2.6 (1.7–4.1) <0.001 Median c c High 110 (34.9%) 177 (49.7%) 1.0 d d Low 205 (65.1%) 179 (50.3%) 1.9 (1.4–2.6) <0.001 Stratification by HCC risk factors including age (≤50 years, >50 years), sex (female, male), alcohol drinking (never, ever), cigarette smoking (never, ever), HBsAg status (negative, positive), anti-HCV status (negative, positive), and HCC family history (no, yes), produced comparable results, regardless of quartile or median LINE-1 methylation evaluated (data not shown). Additional analyses showed that the interactions between LINE-1 methylation and these HCC risk factors in relation to HCC risk were not statistically significant (data not shown). No significant associations were observed of age, alcohol drinking, cigarette smoking, HBsAg status, anti-HCV status or HCC family history with LINE-1 methylation, based on control subjects. However, males (79.8% 5mC) had higher LINE-1 methylation than females (78.2% 5mC; P =0.02) among controls, consistent with previous findings (El-Maarri et al., 2007 2007 ; Hsiung et al., 2007 2007 ). No significant correlation was found between LINE-1 methylation and tumor size, Edmondson-Steiner grade, or tumor stage (data not shown). Discussion 1 Global hypomethylation in blood leukocyte DNA has been reported to correlate with increased risk for several types of cancer (Pufulete et al., 2003 2003 ; Hsiung et al., 2007 2007 ; Moore et al., 2008 2008 ; Choi et al., 2009 2009 ; Cho et al., 2010 2010 ; Mirabello et al., 2010 2010 ). In the present study, we demonstrated for the first time that individuals with global hypomethylation measured in LINE-1 repeats in leukocyte DNA had an increased HCC risk. Taken together, these findings support the idea that there exist common epigenetic basis for the pathogenesis of these different cancers. DNA methylation is a reversible epigenetic mechanism and DNA methylation patterns can be changed in response to exposure to exogenous and endogenous factors (Hsieh, 2000 2000 ; Gluckman et al., 2008 2008 ). Global hypomethylation in leukocyte DNA has been reported to be associated with exposure to risk factors for carcinogenesis (Bollati et al., 2007 2007 ; Rusiecki et al., 2008 2008 ; Breton et al., 2009 2009 ; Liu et al., 2010 2010 ; Wan et al., 2010 2010 ), suggesting that leukocyte DNA hypomethylation may reflect cumulative effects from carcinogenic exposures. On the other hand, global methylation change has been indicated to be partly under genetic control (Bjornsson et al., 2008 2008 ; Hillemacher et al., 2008 2008 ), suggesting that DNA hypomethylation in leukocyte DNA may also reflect transgenerational risks for common human diseases including cancers (Bjornsson et al., 2004 2004 ). Thus, global methylation levels in leukocyte DNA could provide a useful biomarker of susceptibility to certain cancer types. Global DNA hypomethylation has been proposed to contribute to activation of oncogenes and genomic instability in cancer tissues, thereby causing aberrant activation of a wide spectrum of genes, formation of abnormal chromosomal structures and increased mutation rates that convey various growth advantages (Gaudet et al., 2003 2003 ; Feinberg et al., 2006 2006 ). LINE-1 hypomethylation is often found in association with hypermethylation of specific genes in cancer tissues (Cho et al., 2007 2007 ; Ogino et al., 2008 2008 ). Thus, global DNA hypomethylation may also contribute to carcinogenesis by inactivating tumor suppression genes. Moreover, LINE-1 methylation status by itself is likely to have biological effects, which may contribute directly to carcinogenesis through new retrotransposon insertions in human genomes (Iskow et al., 2010 2010 ). Global DNA hypomethylation has been demonstrated to lead to the transcriptional activation of LINE-1 promoters, which can alter the transcriptome both in primary bladder tumors and in their premalignant urothelium counterparts (Wolff et al., 2010 2010 ). These findings suggested that hypomethylation of LINE-1 may play a role not only in cancer but also in cancer predisposition. Our study had the advantages of being based on a relatively large sample size, careful matching of controls to HCC cases by age, sex and HBsAg status, and pyrosequencing-based quantitative analysis which produced individual measures of methylation at three CpG dinucleotide positions, thus more accurately reflecting DNA methylation in the region. Several limitations in the present study should be noted. Firstly, this was a hospital-based case-control study, which may result in selection bias of participants. Moreover, given that DNA methylation in cancer patients was measured after cancer diagnosis and DNA hypomethylation could be detected in sera as well as HCC cells in peripheral blood of HCC cases (Tangkijvanich et al., 2007 2007 ; Lee et al., 2009 2009 ), blood DNA hypomethylation in cancer patients we observed may partly represent the methylation level of DNA derived from HCC cells. As degree of hypomethylation in tumor cells and number of HCC cells in circulating peripheral blood progress according to increased tumor stage, further analyses of LINE-1 methylation in relation to HCC risk were repeated using cases only with early stage of tumor (Stage I/II), which showed no major differences in risk estimates from the results based on the total number of cases (data not shown). In this context, the potentially-confounding effect of contaminating DNA from HCC cells on the association between LINE-1 hypomethylation and HCC risk observed in the present study would be minimal. However, further prospective studies using HCC-free subjects are needed to verify the risk-effect of global hypomethylation in blood DNA on HCC development. Secondly, information on diet was not available here, which may also influence methylation levels in genomic DNA (Suzuki et al., 2006 2006 ; Schernhammer et al., 2010 2010 ). Lastly, although LINE-1 repetitive elements have been used as a surrogate for global DNA methylation content, its methylation level is not equivalent to global DNA methylation content (Yang et al., 2004 2004 ; Choi et al., 2009 2009 ). Moreover, PCR primers were designed towards a consensus LINE-1 sequence and allowed for the amplification of only a small pool of LINE-1 repetitive elements dispersed throughout the genome (Yang et al., 2004 2004 ). In conclusion, global hypomethylation measured in LINE-1 repeats in blood leukocyte DNA shows an increased risk for HCC. Our data provide the evidence that global hypomethylation detected in the easily obtainable DNA source of blood leukocytes may help identify individuals at risk for HCC. References HT Bjornsson , MD Fallin , AP Feinberg . An integrated epigenetic and genetic approach to common human disease . Trends Genet , 2004 . 20 ( 8 ): 350 - 358 . 15262407 DOI: 10.1016/j.tig.2004.06.009 http://doi.org/10.1016/j.tig.2004.06.009 . An integrated epigenetic and genetic approach to common human disease HT Bjornsson , MI Sigurdsson , MD Fallin , RA Irizarry , T Aspelund , H Cui , W Yu , MA Rongione , TJ Ekstrom , TB Harris , 等 . Intra-individual change over time in DNA methylation with familial clustering . JAMA , 2008 . 299 ( 24 ): 2877 - 2883 . 18577732 DOI: 10.1001/jama.299.24.2877 http://doi.org/10.1001/jama.299.24.2877 . Intra-individual change over time in DNA methylation with familial clustering V Bollati , A Baccarelli , L Hou , M Bonzini , S Fustinoni , D Cavallo , HM Byun , J Jiang , B Marinelli , AC Pesatori , 等 . Changes in DNA methylation patterns in subjects exposed to low-dose benzene . Cancer Res , 2007 . 67 ( 3 ): 876 - 880 . 17283117 DOI: 10.1158/0008-5472.CAN-06-2995 http://doi.org/10.1158/0008-5472.CAN-06-2995 . Changes in DNA methylation patterns in subjects exposed to low-dose benzene CV Breton , HM Byun , M Wenten , F Pan , A Yang , FD Gilliland . Prenatal tobacco smoke exposure affects global and gene-specific DNA methylation . Am J Respir Crit Care Med , 2009 . 180 ( 5 ): 462 - 467 . 19498054 DOI: 10.1164/rccm.200901-0135OC http://doi.org/10.1164/rccm.200901-0135OC . Prenatal tobacco smoke exposure affects global and gene-specific DNA methylation AF Burden , NC Manley , AD Clark , SM Gartler , CD Laird , RS Hansen . Hemimethylation and non-CpG methylation levels in a promoter region of human LINE-1 (L1) repeated elements . J Biol Chem , 2005 . 280 ( 15 ): 14413 - 14419 . 15710616 DOI: 10.1074/jbc.M413836200 http://doi.org/10.1074/jbc.M413836200 . Hemimethylation and non-CpG methylation levels in a promoter region of human LINE-1 (L1) repeated elements NY Cho , BH Kim , M Choi , EJ Yoo , KC Moon , YM Cho , D Kim , GH Kang . Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features . J Pathol , 2007 . 211 ( 3 ): 269 - 277 . 17139617 DOI: 10.1002/path.2106 http://doi.org/10.1002/path.2106 . Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features YH Cho , H Yazici , HC Wu , MB Terry , K Gonzalez , M Qu , N Dalay , RM Santella . Aberrant promoter hypermethylation and genomic hypomethylation in tumor, adjacent normal tissues and blood from breast cancer patients . Anticancer Res , 2010 . 30 ( 7 ): 2489 - 2496 . 20682973 . Aberrant promoter hypermethylation and genomic hypomethylation in tumor, adjacent normal tissues and blood from breast cancer patients JY Choi , SR James , PA Link , SE McCann , CC Hong , W Davis , MK Nesline , CB Ambrosone , AR Karpf . Association between global DNA hypomethylation in leukocytes and risk of breast cancer . Carcinogenesis , 2009 . 30 ( 11 ): 1889 - 1897 . 19584139 DOI: 10.1093/carcin/bgp143 http://doi.org/10.1093/carcin/bgp143 . Association between global DNA hypomethylation in leukocytes and risk of breast cancer R Cordaux , MA Batzer . The impact of retrotransposons on human genome evolution . Nat Rev Genet , 2009 . 10 ( 10 ): 691 - 703 . 19763152 DOI: 10.1038/nrg2640 http://doi.org/10.1038/nrg2640 . The impact of retrotransposons on human genome evolution PM Das , R Singal . DNA methylation and cancer . J Clin Oncol , 2004 . 22 ( 22 ): 4632 - 4642 . 15542813 DOI: 10.1200/JCO.2004.07.151 http://doi.org/10.1200/JCO.2004.07.151 . DNA methylation and cancer O El-Maarri , T Becker , J Junen , SS Manzoor , A Diaz-Lacava , R Schwaab , T Wienker , J Oldenburg . Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males . Hum Genet , 2007 . 122 ( 5 ): 505 - 514 . 17851693 DOI: 10.1007/s00439-007-0430-3 http://doi.org/10.1007/s00439-007-0430-3 . Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males AP Feinberg , B Tycko . The history of cancer epigenetics . Nat Rev Cancer , 2004 . 4 ( 2 ): 143 - 153 . 14732866 DOI: 10.1038/nrc1279 http://doi.org/10.1038/nrc1279 . The history of cancer epigenetics AP Feinberg , R Ohlsson , S Henikoff . The epigenetic progenitor origin of human cancer . Nat Rev Genet , 2006 . 7 ( 1 ): 21 - 33 . 16369569 DOI: 10.1038/nrg1748 http://doi.org/10.1038/nrg1748 . The epigenetic progenitor origin of human cancer EJ Formeister , M Tsuchiya , H Fujii , S Shpyleva , IP Pogribny , I Rusyn . Comparative analysis of promoter methylation and gene expression endpoints between tumorous and non-tumorous tissues from HCV-positive patients with hepatocellular carcinoma . Mutat Res , 2010 . 692 ( 1-2 ): 26 - 33 . 20736025 DOI: 10.1016/j.mrfmmm.2010.07.013 http://doi.org/10.1016/j.mrfmmm.2010.07.013 . Comparative analysis of promoter methylation and gene expression endpoints between tumorous and non-tumorous tissues from HCV-positive patients with hepatocellular carcinoma F Gaudet , JG Hodgson , A Eden , L Jackson-Grusby , J Dausman , JW Gray , H Leonhardt , R Jaenisch . Induction of tumors in mice by genomic hypomethylation . Science , 2003 . 300 ( 5618 ): 489 - 492 . 12702876 DOI: 10.1126/science.1083558 http://doi.org/10.1126/science.1083558 . Induction of tumors in mice by genomic hypomethylation PD Gluckman , MA Hanson , C Cooper , KL Thornburg . Effect of in utero and early-life conditions on adult health and disease . N Engl J Med , 2008 . 359 ( 1 ): 61 - 73 . 18596274 DOI: 10.1056/NEJMra0708473 http://doi.org/10.1056/NEJMra0708473 . Effect of in utero and early-life conditions on adult health and disease AI Gomaa , SA Khan , MB Toledano , I Waked , SD Taylor-Robinson . Hepatocellular carcinoma: epidemiology, risk factors and pathogenesis . World J Gastroenterol , 2008 . 14 ( 27 ): 4300 - 4308 . 18666317 DOI: 10.3748/wjg.14.4300 http://doi.org/10.3748/wjg.14.4300 . Hepatocellular carcinoma: epidemiology, risk factors and pathogenesis T Hillemacher , H Frieling , S Moskau , MA Muschler , A Semmler , J Kornhuber , T Klockgether , S Bleich , M Linnebank . Global DNA methylation is influenced by smoking behaviour . Eur Neuropsychopharmacol , 2008 . 18 ( 4 ): 295 - 298 . 18242065 DOI: 10.1016/j.euroneuro.2007.12.005 http://doi.org/10.1016/j.euroneuro.2007.12.005 . Global DNA methylation is influenced by smoking behaviour CL Hsieh . Dynamics of DNA methylation pattern . Curr Opin Genet Dev , 2000 . 10 ( 2 ): 224 - 228 . 10753782 DOI: 10.1016/S0959-437X(00)00064-2 http://doi.org/10.1016/S0959-437X(00)00064-2 . Dynamics of DNA methylation pattern DT Hsiung , CJ Marsit , EA Houseman , K Eddy , CS Furniss , MD McClean , KT Kelsey . Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma . Cancer Epidemiol Biomarkers Prev , 2007 . 16 ( 1 ): 108 - 114 . 17220338 DOI: 10.1158/1055-9965.EPI-06-0636 http://doi.org/10.1158/1055-9965.EPI-06-0636 . Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma RC Iskow , MT McCabe , RE Mills , S Torene , WS Pittard , AF Neuwald , EG van Meir , PM Vertino , SE Devine . Natural mutagenesis of human genomes by endogenous retrotransposons . Cell , 2010 . 141 ( 7 ): 1253 - 1261 . 20603005 DOI: 10.1016/j.cell.2010.05.020 http://doi.org/10.1016/j.cell.2010.05.020 . Natural mutagenesis of human genomes by endogenous retrotransposons YJ Kim , HS Lee . Single nucleotide polymorphisms associated with hepatocellular carcinoma in patients with chronic hepatitis B virus infection . Intervirology , 2005 . 48 ( 1 ): 10 - 15 . 15785084 DOI: 10.1159/000082089 http://doi.org/10.1159/000082089 . Single nucleotide polymorphisms associated with hepatocellular carcinoma in patients with chronic hepatitis B virus infection ES Lander , LM Linton , B Birren , C Nusbaum , MC Zody , J Baldwin , K Devon , K Dewar , M Doyle , W FitzHugh , 等 . Initial sequencing and analysis of the human genome . Nature , 2001 . 409 ( 6822 ): 860 - 921 . 11237011 DOI: 10.1038/35057062 http://doi.org/10.1038/35057062 . Initial sequencing and analysis of the human genome HS Lee , BH Kim , NY Cho , EJ Yoo , M Choi , SH Shin , JJ Jang , KS Suh , YS Kim , GH Kang . Prognostic implications of and relationship between CpG island hypermethylation and repetitive DNA hypomethylation in hepatocellular carcinoma . Clin Cancer Res , 2009 . 15 ( 3 ): 812 - 820 . 19188151 DOI: 10.1158/1078-0432.CCR-08-0266 http://doi.org/10.1158/1078-0432.CCR-08-0266 . Prognostic implications of and relationship between CpG island hypermethylation and repetitive DNA hypomethylation in hepatocellular carcinoma HJ Lei , GY Chau , WY Lui , SH Tsay , KL King , CC Loong , CW Wu . Prognostic value and clinical relevance of the 6th Edition 2002 American Joint Committee on Cancer staging system in patients with resectable hepatocellular carcinoma . J Am Coll Surg , 2006 . 203 ( 4 ): 426 - 435 . 17000385 DOI: 10.1016/j.jamcollsurg.2006.06.030 http://doi.org/10.1016/j.jamcollsurg.2006.06.030 . Prognostic value and clinical relevance of the 6th Edition 2002 American Joint Committee on Cancer staging system in patients with resectable hepatocellular carcinoma U Lim , A Flood , SW Choi , D Albanes , AJ Cross , A Schatzkin , R Sinha , HA Katki , B Cash , P Schoenfeld , 等 . Genomic methylation of leukocyte DNA in relation to colorectal adenoma among asymptomatic women . Gastroenterology , 2008 . 134 ( 1 ): 47 - 55 . 18166347 DOI: 10.1053/j.gastro.2007.10.013 http://doi.org/10.1053/j.gastro.2007.10.013 . Genomic methylation of leukocyte DNA in relation to colorectal adenoma among asymptomatic women F Liu , JK Killian , M Yang , RL Walker , JA Hong , M Zhang , S Davis , Y Zhang , M Hussain , S Xi , 等 . Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate . Oncogene , 2010 . 29 ( 25 ): 3650 - 3664 . 20440268 DOI: 10.1038/onc.2010.129 http://doi.org/10.1038/onc.2010.129 . Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate L Mirabello , SA Savage , L Korde , SM Gadalla , MH Greene . LINE-1 methylation is inherited in familial testicular cancer kindreds . BMC Med Genet , 2010 . 11 ( 1 ): 77 20478068 DOI: 10.1186/1471-2350-11-77 http://doi.org/10.1186/1471-2350-11-77 . LINE-1 methylation is inherited in familial testicular cancer kindreds LE Moore , RM Pfeiffer , C Poscablo , FX Real , M Kogevinas , D Silverman , R Garcia-Closas , S Chanock , A Tardon , C Serra , 等 . Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish bladder cancer study: a case-control study . Lancet Oncol , 2008 . 9 ( 4 ): 359 - 366 . 18339581 DOI: 10.1016/S1470-2045(08)70038-X http://doi.org/10.1016/S1470-2045(08)70038-X . Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish bladder cancer study: a case-control study TR Morgan , S Mandayam , MM Jamal . Alcohol and hepatocellular carcinoma . Gastroenterology , 2004 . 127 ( 5 ): S87 - S96 . 15508108 DOI: 10.1053/j.gastro.2004.09.020 http://doi.org/10.1053/j.gastro.2004.09.020 . Alcohol and hepatocellular carcinoma S Ogino , T Kawasaki , K Nosho , M Ohnishi , Y Suemoto , GJ Kirkner , CS Fuchs . LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer . Int J Cancer , 2008 . 122 ( 12 ): 2767 - 2773 . 18366060 DOI: 10.1002/ijc.23470 http://doi.org/10.1002/ijc.23470 . LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer H Okuda . Hepatocellular carcinoma development in cirrhosis . Best Pract Res Clin Gastroenterol , 2007 . 21 ( 1 ): 161 - 173 . 17223503 DOI: 10.1016/j.bpg.2006.07.002 http://doi.org/10.1016/j.bpg.2006.07.002 . Hepatocellular carcinoma development in cirrhosis DM Parkin , F Bray , J Ferlay , P Pisani . Global cancer statistics, 2002 . CA Cancer J Clin , 2005 . 55 ( 2 ): 74 - 108 . 15761078 DOI: 10.3322/canjclin.55.2.74 http://doi.org/10.3322/canjclin.55.2.74 . Global cancer statistics, 2002 M Pufulete , R Al-Ghnaniem , AJ Leather , P Appleby , S Gout , C Terry , PW Emery , TA Sanders . Folate status, genomic DNA hypomethylation, and risk of colorectal adenoma and cancer: a case control study . Gastroenterology , 2003 . 124 ( 5 ): 1240 - 1248 . 12730865 DOI: 10.1016/S0016-5085(03)00279-8 http://doi.org/10.1016/S0016-5085(03)00279-8 . Folate status, genomic DNA hypomethylation, and risk of colorectal adenoma and cancer: a case control study JA Rusiecki , A Baccarelli , V Bollati , L Tarantini , LE Moore , EC Bonefeld-Jorgensen . Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit . Environ Health Perspect , 2008 . 116 ( 11 ): 1547 - 1552 . 19057709 DOI: 10.1289/ehp.11338 http://doi.org/10.1289/ehp.11338 . Global DNA hypomethylation is associated with high serum-persistent organic pollutants in Greenlandic Inuit ES Schernhammer , E Giovannucci , T Kawasaki , B Rosner , CS Fuchs , S Ogino . Dietary folate, alcohol and B vitamins in relation to LINE-1 hypomethylation in colon cancer . Gut , 2010 . 59 ( 6 ): 794 - 799 . 19828464 DOI: 10.1136/gut.2009.183707 http://doi.org/10.1136/gut.2009.183707 . Dietary folate, alcohol and B vitamins in relation to LINE-1 hypomethylation in colon cancer MI Shariff , IJ Cox , AI Gomaa , SA Khan , W Gedroyc , SD Taylor-Robinson . Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics . Expert Rev Gastroenterol Hepatol , 2009 . 3 ( 4 ): 353 - 367 . 19673623 DOI: 10.1586/egh.09.35 http://doi.org/10.1586/egh.09.35 . Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics H Suzuki , M Toyota , H Sato , T Sonoda , F Sakauchi , M Mori . Roles and causes of abnormal DNA methylation in gastrointestinal cancers . Asian Pac J Cancer Prev , 2006 . 7 ( 2 ): 177 - 185 . 16839207 . Roles and causes of abnormal DNA methylation in gastrointestinal cancers P Tangkijvanich , N Hourpai , P Rattanatanyong , N Wisedopas , V Mahachai , A Mutirangura . Serum LINE-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma . Clin Chim Acta , 2007 . 379 ( 1-2 ): 127 - 133 . 17303099 DOI: 10.1016/j.cca.2006.12.029 http://doi.org/10.1016/j.cca.2006.12.029 . Serum LINE-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma L Tarantini , M Bonzini , P Apostoli , V Pegoraro , V Bollati , B Marinelli , L Cantone , G Rizzo , L Hou , J Schwartz , 等 . Effects of particulate matter on genomic DNA methylation content and iNOS promoter methylation . Environ Health Perspect , 2009 . 117 ( 2 ): 217 - 222 . 19270791 DOI: 10.1289/ehp.11898 http://doi.org/10.1289/ehp.11898 . Effects of particulate matter on genomic DNA methylation content and iNOS promoter methylation GA Thomas , ED Williams . Production of thyroid tumours in mice by demethylating agents . Carcinogenesis , 1992 . 13 ( 6 ): 1039 - 1042 . 1376218 DOI: 10.1093/carcin/13.6.1039 http://doi.org/10.1093/carcin/13.6.1039 . Production of thyroid tumours in mice by demethylating agents YJ Wan , YY Li , W Xia , J Chen , ZQ Lv , HC Zeng , L Zhang , WJ Yang , T Chen , Y Lin , 等 . Alterations in tumor biomarker GSTP gene methylation patterns induced by prenatal exposure to PFOS . Toxicology , 2010 . 274 ( 1-3 ): 57 - 64 . 20621739 DOI: 10.1016/j.tox.2010.05.006 http://doi.org/10.1016/j.tox.2010.05.006 . Alterations in tumor biomarker GSTP gene methylation patterns induced by prenatal exposure to PFOS DJ Weisenberger , M Campan , TI Long , M Kim , C Woods , E Fiala , M Ehrlich , PW Laird . Analysis of repetitive element DNA methylation by MethyLight . Nucleic Acids Res , 2005 . 33 ( 21 ): 6823 - 6836 . 16326863 DOI: 10.1093/nar/gki987 http://doi.org/10.1093/nar/gki987 . Analysis of repetitive element DNA methylation by MethyLight MJ Wilson , N Shivapurkar , LA Poirier . Hypomethylation of hepatic nuclear DNA in rats fed with a carcinogenic methyl-deficient diet . Biochem J , 1984 . 218 ( 3 ): 987 - 990 . 6721844 . Hypomethylation of hepatic nuclear DNA in rats fed with a carcinogenic methyl-deficient diet EM Wolff , HM Byun , HF Han , S Sharma , PW Nichols , KD Siegmund , AS Yang , PA Jones , G Liang . Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer . PLoS Genet , 2010 . 6 ( 4 ): e1000917 20421991 DOI: 10.1371/journal.pgen.1000917 http://doi.org/10.1371/journal.pgen.1000917 . Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer AS Yang , MR Estecio , K Doshi , Y Kondo , EH Tajara , JP Issa . A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements . Nucleic Acids Res , 2004 . 32 ( 3 ): e38 14973332 DOI: 10.1093/nar/gnh032 http://doi.org/10.1093/nar/gnh032 . A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements ZZ Zhu , D Sparrow , L Hou , L Tarantini , V Bollati , AA Litonjua , A Zanobetti , P Vokonas , RO Wright , A Baccarelli , 等 . Repetitive element hypomethylation in blood leukocyte DNA and cancer incidence, prevalence, and mortality in elderly individuals: the normative aging study . Cancer Causes Control , 2011 . 22 ( 3 ): 437 - 447 . 21188491 DOI: 10.1007/s10552-010-9715-2 http://doi.org/10.1007/s10552-010-9715-2 . Repetitive element hypomethylation in blood leukocyte DNA and cancer incidence, prevalence, and mortality in elderly individuals: the normative aging study 10.1631/jzus.B1000422 2010-12-05 2011-09-12 2011-07-27 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 12