Global changes of 5-hydroxymethylcytosine and 5-methylcytosine from normal to tumor tissues are associated with carcinogenesis and prognosis in colorectal cancer

Global changes of 5-hydroxymethylcytosine and 5-methylcytosine from normal to tumor tissues are associated with carcinogenesis and prognosis in colorectal cancer 正常组织与肿瘤组织中总体5hmC和5mC水平的变化程度与结直肠癌肿瘤发生及预后有关 1 2 3 first-author 4 5 1 2 1 2 6 1 2 7 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:zhuym@zju.edu.cnlmd@zju.edu.cn †E-mail:zhuym@zju.edu.cnlmd@zju.edu.cn zhuym@zju.edu.cn 1 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:zhuym@zju.edu.cnlmd@zju.edu.cn †E-mail:zhuym@zju.edu.cnlmd@zju.edu.cn zhuym@zju.edu.cn Department of Pathology, School of Medicine, Zhejiang University, Hangzhou 310058, China Department of Pathology, School of Medicine, Zhejiang University, Hangzhou 310058, China Key Laboratory of Disease Proteomics of Zhejiang Province, Hangzhou 310058, China Key Laboratory of Disease Proteomics of Zhejiang Province, Hangzhou 310058, China Pathology Department, Zhejiang Cancer Hospital, Hangzhou 310022, China Pathology Department, Zhejiang Cancer Hospital, Hangzhou 310022, China Medical Research Center /Human Tissue Bank, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, Linhai 317000, China Medical Research Center /Human Tissue Bank, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, Linhai 317000, China Department of Pathology, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, Linhai 317000, China Department of Pathology, Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, Linhai 317000, China Department of Urology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Urology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Epidemiology and Biostatistics, School of Public Health, Zhejiang University, Hangzhou 310058, China Department of Epidemiology and Biostatistics, School of Public Health, Zhejiang University, Hangzhou 310058, China Aberrant DNA methylation has raised widespread attention in tumorigenesis. In this study, we aimed to investigate the changes of global DNA methylation and hydroxymethylation from normal to tumor tissues in colorectal cancer (CRC) and their association with the prognosis. The levels of genomic 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in cancerous tissues were significantly lower than those in corresponding adjacent normal tissues. The genomic levels of 5mC were significantly positively correlated with 5hmC in normal and cancerous tissues (all P < 0.05). The ratio of 5mC in cancerous tissues to matched normal tissues (C/N-5mC) was also significantly positively correlated with the ratio of 5hmC in cancerous tissues to matched normal tissues (C/N-5hmC) ( P =0.01). The 5mC levels and C/N-5mC ratios decreased with age (all P < 0.05). Higher 5mC and 5hmC levels were found in rectal than in colon tissues (all P < 0.05). High levels of 5mC in cancerous tissues and high C/N-5hmC ratios were each associated with lymph node metastasis (all P < 0.05). Survival analysis indicated that the C/N-5mC ratio ( P =0.04) is an independent protective factor for overall survival. The data showed that patients with a combination of high C/N-5hmC and low C/N-5mC ratios tended to have a worse prognosis ( P < 0.01). Our findings showed that the C/N-5mC ratio may be an independent prognostic factor for CRC outcome. Patients with both a high C/N-5hmC ratio and a low C/N-5mC ratio exhibited the worst survival, suggesting that 5mC and 5hmC can be used as critical markers in tumorigenesis and prognosis estimation. 目的 2 研究DNA总体甲基化、羟甲基化水平与结直肠癌发生、发展及预后的关系。 创新点 2 采用肿瘤与正常组织总体5-甲基胞嘧啶（5mC）、5-羟甲基胞嘧啶（5hmC）的比值并探讨其与结直肠癌临床病理变化和预后的关系。 方法 2 采用酶联免疫吸附试验（ELISA）方法检测肿瘤与相应正常组织基因组中总体5hmC和5mC水平。 结论 2 C/N-5mC比值的水平对结直肠癌的预后是一个独立的保护性因子（ P =0.04）。当病人同时具有高水平的C/N-5hmC和低水平的C/N-5mC，其预后更差（ P < 0.01）。因此，可以使用5mC和5hmC对结直肠癌的发生和预后进行评估。 5-Hydroxymethylcytosine (5hmC) 5-Methylcytosine (5mC) Prognosis Colorectal cancer 5-羟甲基胞嘧啶 5-甲基胞嘧啶 预后 结直肠癌 Introduction 1 Over recent years, colorectal cancer (CRC) has been well-studied. It has also become one of the most common cancers. Its pathogenesis is characteristic of sequential genetic and epigenetic changes (van Engeland et al., 2011 2011 ). Epigenetic modification denotes heritable traits of gene expression that do not include alterations of the DNA sequences. Epigenetic changes mainly affect chromatin structure stability, gene expression, tissue-specific modulation, genome integrity, genomic imprinting, embryonic development, and X-chromosome inactivation in females. Epigenetic changes often refer to reversible DNA methylation as well as histone acetylation, chromatin looping, and small noncoding RNAs (Turek-Plewa and Jagodzinski, 2005 2005 ). Aberrant DNA methylation occurs in many different kinds of cancer and plays a key role in tumorigenesis. In many human cancers, global genomic hypomethylation as well as hypermethylation in promoter regions has been extensively studied. These alterations lead to genomic instability and gene regression or even silencing (Bird, 2002 2002 ; Robertson, 2005 2005 ; Baylin and Jones, 2011 2011 ; Dawson and Kouzarides, 2012 2012 ). In CRC, low genomic 5-methylcytosine (5mC) was first found in tumor tissues in 1988 (Feinberg et al., 1988 1988 ). Global DNA demethylation is age-dependent, even preceding the process of carcinogenesis (Suzuki et al., 2006 2006 ). Previous findings have shown that lower levels of 5mC correlate well with advanced malignancy grade in breast cancer and colon cancer (Barciszewska et al., 2007 2007 ). Some studies have shown that the Line-1 (long interspersed nuclear element 1) methylation level, which may represent global methylation status, is negatively associated with T-stage and is significantly reduced in lymph node metastases in CRC (Sunami et al., 2011 2011 ; Morikawa et al., 2012 2012 ). Furthermore, early studies found that 5mC is an independent marker of poor survival outcome in CRC patients (Frigola et al., 2005 2005 ; Ogino et al., 2008 2008 ; Li et al., 2014 2014 ). However, there are few studies analyzing the relationship between clinicopathological features and the degree of change in 5mC from normal to tumor tissues in CRC patients. Kriaucionis and Heintz ( 2009 2009 ) used the technologies of high-pressure liquid chromatography, thin-layer chromatography, and mass spectrometry to identify 5-hydroxymethylcytosine (5hmC) in the brain. Another group reported that ten eleven translocation (TET) can convert 5mC to 5hmC in cultured cells and in vitro (Sun et al., 2013 2013 ). Just like 5mC, 5hmC exists ubiquitously and stably in human tissues, and its reduction in cancerous tissues has been reported in a variety of malignancies including hematological malignancies and solid tumors, as well as in CRC (Robertson, 2005 2005 ; Jin et al., 2011 2011 ; Dawson and Kouzarides, 2012 2012 ; Zhang et al., 2013 2013 ; Wang et al., 2014 2014 ). A recent study of acute myeloid leukemia showed that high levels of 5hmC are correlated with inferior overall survival (Kroeze et al., 2014 2014 ). However, in melanoma, a reduced level of 5hmC is a marker of worse prognosis and is associated with dysplastic cytomorphological features and tumor progression (Larson et al., 2014 2014 ). In solid tumors, low 5hmC levels represent poor overall survival and high cumulative recurrence (Liu et al., 2014 2014 ). In addition, 5hmC levels have also been highly correlated with tumor stage (Chen et al., 2013 2013 ). So far, studies have reported that 5hmC has its own additional biological function as an intermediate base in DNA demethylation (Pfeifer et al., 2013 2013 ). Ficz et al. ( 2011 2011 ) found that 5hmC in the promoter regions is associated with an increased transcriptional level. Also, Robertson et al. ( 2011 2011 ) reported that the presence of 5hmC at the promoter negatively regulates gene expression. So the exact function of 5hmC is still unclear. A recent study of CRC indicated that 5hmC in promoters resists DNA hypermethylation and highlighted that 5hmC plays an important role in cancerous cell proliferation (Uribe-Lewis et al., 2015 2015 ). Therefore, given the potential roles of 5mC and 5hmC in carcinogenesis, we explored their association with clinicopathological parameters and outcomes in CRC patients. Materials and methods 1 Participants 2 From 2006 to 2012, 71 CRC patients (40 men and 31 women) were recruited from Taizhou Hospital in Zhejiang Province, China. Primary cancerous colorectal tissues and corresponding adjacent normal tissues from the surgical margin were collected after surgery. No patients received radiotherapy or chemotherapy before surgery. The diagnosis of CRC was made by senior pathologists. The pathological features and clinical stages of primary tumors were defined on the basis of the criteria of the American Joint Commission on Cancer/International Union Against Cancer (AJCC/UICC). Patients were followed up until death or censored at five years from the date of surgery. All the tissues in this study, including tumor tissues and corresponding normal tissues, were the primary tissues. No metastasis tissue was analyzed in this study. However, we compared the difference between patients with and without lymph node metastasis or distant metastasis. The protocol was approved by the Research Ethics Committee of the School of Medicine, Zhejiang University, Hangzhou, China. Genomic DNA extraction 2 Genomic DNA was extracted from all 142 samples (cancerous and adjacent normal tissues) using an E.Z.N.A. ® DNA/RNA isolation kit (Omega Bio-Tek, Norcross, GA, USA) and quantified using a spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA). Determination of genomic 5hmC and 5mC levels 2 Genomic 5hmC and 5mC levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (MethylFlash Hydroxymethylated/Methylated DNA Quantification Kit (Fluorometric); Epigentek, Farmingdale, NY, USA). Genomic DNA (100 ng) was used to assess the levels of 5hmC and 5mC, following the manufacturer’s protocols (Li and Liu, 2011 2011 ). The percentages of 5mC (5mC%) and 5hmC (5hmC%) in total DNA were calculated using the following formulae: 5mC%=(OD sample −OD negative )/(slope × 2 ×S ) × 100%, 5hmC%=(OD sample −OD negative )/(slope × 5 ×S ) × 100%, where OD sample and OD negative are the optical densities of sample and negative control, respectively. S is the amount of input sample DNA in nanograms. Statistical analysis 2 SPSS 19.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. For variables with non-normal distributions, medians and interquartile ranges are presented. The Wilcoxon signed-rank test for paired non-parametric data was used to determine the differences in 5mC and 5hmC levels between cancerous and adjacent normal tissues in overall and subgroup analysis. Correlations between 5hmC and 5mC levels were evaluated by the Spearman’s rank correlation coefficient. The associations between 5hmC and 5mC levels and clinicopathological parameters were calculated using the Mann-Whitney test. The continuous variables, 5mC and 5hmC levels, were classified into categorical variables according to the receiver operating characteristic curves combined with the median value. The cut-off values were the points at the base of the maximal Youden index (sensitivity+specificity–1) (Liang et al., 2015 2015 ). Kaplan-Meier survival analysis was used to calculate the overall survival time, and prognostic differences were compared with the log-rank test. The adjusted hazard ratio (HR) was measured using the multivariate Cox proportional hazard model. Statistical significance was set at P < 0.05 (two-tailed). Graphs were created using GraphPad Prism 5 (San Diego, CA, USA). Results 1 Basic characteristics of participants 2 The demographic characteristics of the 71 patients are summarized in Table 1 1 . The age at diagnosis ranged from 28 to 84 years (median, 64 years); 37 patients (52.1%) had colon cancer and the others had rectal cancer; 38 had lymph node metastasis and 8 had distant metastasis; in 35, the tumor had penetrated to the visceral peritoneum or other organs; and the 5-year overall survival rate of the cohort was 60.6%. All cases were classified according to the 7th edition of the pathologic tumor-node-metastasis (pTNM) classification of the AJCC/UICC. Data are expressed as median (P25–P75); P a values were determined with the Mann-Whitney test, and P b values with the Wilcoxon signed-rank test 2 Variable 2 Number 4 Level of 5mC (%) 4 Level of 5hmC (%) Normal tissues Cancerous tissues P b C/N Normal tissues Cancerous tissues P b C/N 2 Age (year)  ≤60 30 6.58 (4.59–8.80) 5.32 (4.18–7.52) <0.01 0.86 (0.63–1.07) 0.07 (0.03–0.11) 0.05 (0.01–0.07) <0.01 0.61 (0.39–0.85)  >60 41 5.81 (4.21–8.02) 4.15 (3.09–5.28) <0.01 0.69 (0.55–0.93) 0.08 (0.03–0.16) 0.04 (0.01–0.12) <0.01 0.53 (0.36–0.83)   P a 0.36 <0.01 0.04 0.58 0.87 0.44 2 Gender  Male 40 5.54 (4.25–8.04) 4.53 (3.46–5.72) <0.01 0.79 (0.63–1.00) 0.07 (0.02–0.15) 0.04 (0.01–0.11) <0.01 0.61 (0.40–0.81)  Female 31 6.71 (4.41–9.16) 4.42 (3.45–5.99) <0.01 0.70 (0.55–1.02) 0.08 (0.05–0.13) 0.05 (0.02–0.08) <0.01 0.53 (0.37–1.02)   P a 0.18 0.98 0.31 0.73 0.54 0.91 3 Location  Colon 37 4.73 (4.11–6.05) 4.03 (3.23–5.20) <0.01 0.83 (0.64–1.05) 0.04 (0.02–0.08) 0.01 (0.01–0.05) <0.01 0.52 (0.29–0.68)  Rectum 34 7.86 (6.45–8.53) 5.05 (4.01–6.42) <0.01 0.69 (0.52–0.95) 0.12 (0.07–0.23) 0.10 (0.04–0.14) <0.01 0.65 (0.48–0.89)   P a <0.01 0.04 0.17 <0.01 <0.01 0.01 3 Tumor-node-metastasis (TNM) stage  I–II 29 5.67 (4.23–7.50) 4.32 (3.40–4.93) <0.01 0.70 (0.53–1.06) 0.10 (0.03–0.19) 0.05 (0.01–0.11) <0.01 0.48 (0.34–0.75)  III–IV 42 6.37 (4.36–8.23) 4.97 (3.49–6.42) <0.01 0.83 (0.63–0.95) 0.07 (0.03–0.11) 0.04 (0.01–0.08) <0.01 0.63 (0.45–0.85)   P a 0.33 0.08 0.69 0.29 0.88 0.11 3 Depth of tumor invasion (T)  T1–T3 36 5.37 (4.18–7.82) 4.61 (3.68–5.95) <0.01 0.88 (0.61–1.07) 0.10 (0.04–0.16) 0.05 (0.01–0.12) <0.01 0.61 (0.45–0.84)  T4 35 6.52 (4.72–8.36) 4.32 (3.11–5.67) <0.01 0.69 (0.54–0.91) 0.07 (0.03–0.11) 0.03 (0.01–0.07) <0.01 0.52 (0.33–0.75)   P a 0.26 0.33 0.05 0.21 0.16 0.30 9 Lymph node metastasis (N)  N0 33 6.12 (4.41–7.94) 4.07 (3.17–4.93) <0.01 0.69 (0.49–1.06) 0.09 (0.04–0.19) 0.05 (0.01–0.12) <0.01 0.48 (0.34–0.75)  N1–N2 38 6.05 (4.21–8.26) 5.05 (3.85–6.73) <0.01 0.83 (0.65–0.98) 0.07 (0.03–0.11) 0.04 (0.01–0.08) <0.01 0.63 (0.48–0.85)   P a 0.62 0.02 0.20 0.21 0.77 0.07 3 Distant metastasis (M)  M0 63 6.12 (4.22–8.09) 4.50 (3.62–5.78) <0.01 0.78 (0.59–1.05) 0.08 (0.03–0.15) 0.05 (0.01–0.10) <0.01 0.56 (0.37–0.83)  M1 8 6.29 (4.84–8.24) 3.72 (2.58–5.86) 0.01 0.75 (0.41–0.89) 0.04 (0.02–0.08) 0.02 (0.01–0.04) 0.09 0.56 (0.41–0.95)   P a 0.72 0.25 0.24 0.16 0.25 0.79 5hmC and 5mC levels in CRC and adjacent normal tissues 2 The overall and subgroup percentage levels of 5mC and 5hmC are presented in Table 1 1 and Fig. 1 1 . The median level (P25–P75) of 5hmC in cancerous tissue was 0.05% (range, 0.01%–0.10%), lower than in normal tissue (0.07% (0.03%–0.13%); P < 0.01). Among the CRC patients, 87.3% (62/71) had lower 5hmC levels in the cancerous tissues than in the normal tissues. These differences were consistent even in stratified analysis by age, gender, CRC location, tumor-node-metastasis (TNM) stage, and depth of tumor invasion, as well as lymph node metastasis and distant metastasis (Table 1 1 ). Percentage levels of 5mC (a) and 5hmC (b) in normal and cancerous tissues. Data are expressed as median (P25–P75) using box-plot and the whiskers on vertical bars show minimum and maximum values. P values were determined by the Wilcoxon signed-rank test As for 5mC, its percentage level in cancerous tissues (4.46% (3.47%–5.75%)) was lower than in normal tissues (6.15% (4.32%–8.16%); P < 0.01). Among the CRC patients, 74.6% (53/71) had lower 5mC levels in cancerous tissues than in normal tissues. Similar decreases in the 5mC levels in cancerous tissues were found in the subgroups (Table 1 1 ). The genomic level of 5mC was significantly positively correlated with 5hmC in both cancerous (Spearman’s ρ =0.25, P =0.03) and normal tissues (Spearman’s ρ =0.36, P < 0.01). The cancerous tissue: normal tissue (C/N) ratio of 5mC was also positively correlated with the C/N ratio of 5hmC (Spearman’s ρ =0.31, P =0.01). Associations of 5mC and 5hmC levels with clinicopathological characteristics in CRC 2 The associations between the 5mC and 5hmC levels and clinicopathological characteristics are shown in Table 1 1 . Patients > 60 years old had lower 5mC levels and C/N-5mC ratios than those ≤60 years old ( P < 0.05). No significant difference was found between male and female patients. In tumor and normal tissues, 5mC and 5hmC levels were higher in rectal cancer than in colon cancer (all P < 0.05). Similar results were found for C/N-5hmC ratios ( P < 0.05). Lower C/N-5mC ratios were found in patients with infiltrative growth (Stage T4) (P=0.05). Those with lymph node metastasis tended to have higher 5mC levels (P=0.02), as well as C/N-5hmC ratios (P=0.07) in tumor tissues. Higher 5mC levels and C/N-5hmC ratios in tumor tissues were found in advanced TNM stages (0.05 < P < 0.1). Associations of 5mC and 5hmC levels with overall survival 2 The overall survival of patients stratified by 5mC and 5hmC levels is presented in Table 2 2 and Fig. 2 2 . Among the 71 patients, the median survival time was 50.7 months (interquartile range, 27.6–58.7 months), and the overall survival rate was 59.7%. According to the receiver operating characteristic curves, the cut-off value was 5.81% for 5mC and 0.07% for 5hmC in normal tissues and 4.51% for 5mC and 0.05% for 5hmC in cancerous tissues. The cut-off value for the C/N ratio was 0.92% for 5mC and 0.48% for 5hmC. Kaplan-Meier analysis revealed that CRC patients with a higher C/N-5mC ratio had a longer survival of 54.0 months (standard error of the mean (SEM)=3.0 months) than those with a lower ratio (mean=42.9 months; SEM=2.9 months; P= 0.01). Shorter survival was found in patients with high levels of 5mC in normal tissues ( P =0.07), high levels of 5hmC in cancerous tissues ( P =0.04), and high C/N-5hmC ratios ( P= 0.08). After adjustment for age, sex, and TNM stage with the multiple Cox regression model, significant associations were found in the C/N-5mC ratios (HR=0.36; 95% confidence interval (CI): 0.13–0.96; P= 0.04) and in the 5mC levels in normal tissues ( P< 0.04). However, neither the 5hmC levels in cancerous tissues nor the C/N-5hmC ratios had a significant correlation with survival rate based on the multiple Cox regression model (all P < 0.05). P a values for survival were determined with the log-rank test, and P b values were measured by multivariate analyses of overall survival (Cox proportional hazards regression model) after adjustment for age, sex, and TNM stage. Data were expressed as mean±SEM Variable Number Survival rate (%) Survival time (month) * * P a HR (95% CI) P b 4 5mC levels in normal tissues  Low 34 70.6 51.5±2.9 1  High 37 51.4 42.5±3.4 0.07 2.29 (1.02–5.16) 0.04 4 5mC levels in cancerous tissues  Low 38 65.8 48.1±3.2 1  High 33 54.5 45.2±3.3 0.39 1.63 (0.73–3.64) 0.23 5 C/N-5mC  Low 46 50.0 42.8±3.0 1  High 25 80.0 54.0±3.0 0.01 0.36 (0.13–0.96) 0.04 4 5hmC levels in normal tissues  Low 36 63.9 48.9±2.9 1  High 35 57.1 44.6±3.6 0.57 1.39 (0.65–2.98) 0.40 4 5hmC levels in cancerous tissues  Low 36 72.2 51.8±2.6 1  High 35 48.6 41.7±3.6 0.04 1.84 (0.84–4.05) 0.13 5 C/N-5hmC  Low 24 75.0 51.5±3.8 1  High 47 53.2 44.3±2.8 0.08 2.01 (0.78–5.41) 0.14 Overall survival of 71 CRC patients in relation to the 5hmC levels in cancerous tissues (a), C/N-5hmC (b), C/N-5mC (c), and risk level of the combined model (d). Survival rates were analyzed using the Kaplan-Meier survival test. P values were calculated using the log-rank test and adjusted P values were assessed by multivariate analyses after adjustment for age, sex, and TNM stage Combined effect of 5mC and 5hmC on survival 2 The combined effect of the C/N-5mC and C/N-5hmC ratios on survival is presented in Table 3 3 and Fig. 2 2 . Based on the different values of the C/N-5mC and C/N-5hmC ratios, the 71 patients were divided into four groups. Comparison of the combined signature analysis for all 71 patients showed a significant correlation with overall survival based on Kaplan-Meier curves ( P< 0.01). Patients with both high C/N-5hmC and low C/N-5mC ratios had a shorter survival of 36.2 months than the others (mean=53.1 months; SEM=2.4 months; P < 0.01; Table 3 3 ). In the multiple Cox regression model with adjustment for age, sex, and TNM stage, patients with high C/N-5hmC and low C/N-5mC ratios also showed a worse survival outcome (HR=3.48, 95% CI: 1.54–7.88; P < 0.01). P a values for survival were determined with the log-rank test, and P b values were measured by multivariate analyses of overall survival (Cox proportional hazards regression model) after adjustment for age, sex, and TNM stage. Data were expressed as mean±SEM Group C/N-5hmC C/N-5mC Number Survival rate (%) Survival time (month) * * P a HR (95% CI) P b  A High Low 27 33.3 36.2±3.8 4 <0.01 1  B Low High 5 80.0 49.6±10.1 0.30 (0.04–2.40) 0.25  C High High 20 80.0 55.1±2.7 0.24 (0.08–0.73) 0.01  D Low Low 19 73.7 52.0±3.9 0.33 (0.12–0.92) 0.03 9 3  B+C+D (low risk) 44 68.2 53.1±2.4 1 3  A (high risk) 27 33.3 36.2±3.8 <0.01 3.48 (1.54–7.88) <0.01 Discussion 1 We found that the 5mC and 5hmC levels were significantly reduced in CRC, and were correlated with some of the clinicopathological features. Furthermore, high C/N-5hmC and low C/N-5mC ratios were associated with an unfavorable outcome in CRC patients. It has been suggested that epigenetic alteration is an early and major event during carcinogenesis, especially DNA-methylated modification. So far, many different methods have been developed to assess global DNA methylation and hydroxymethylation levels, such as bisulfite sequencing, liquid chromatography-mass spectrometry, and methylated/hydroxymethylated DNA immunoprecipitation. In this study, we used ELISA to quantify the genome-wide 5hmC and 5mC levels because of its convenience, cost-effectiveness, and relatively high sensitivity and specificity (Jin et al., 2010 2010 ; 2011 2011 ; Li and Liu, 2011 2011 ; Zhu et al., 2014 2014 ). First, the levels of 5mC and 5hmC differed between cancerous and corresponding adjacent tissues, in line with previous studies (Li and Liu, 2011 2011 ; van Engeland et al., 2011 2011 ; Kudo et al., 2012 2012 ). A significant reduction of 22.39% for 5mC and 44.07% for 5hmC was found in cancerous tissues relative to normal tissues (Feinberg et al., 1988 1988 ). A further reduction of 5hmC was noted in cancerous tissues compared to 5mC ( P< 0.01). Higher levels of 5hmC and 5mC were found in rectal than in colon tissues, supporting the evidence that the distribution of 5hmC and 5mC is tissue-dependent (Li and Liu, 2011 2011 ). These results may be affected by individual heterogeneity and variation, which existed in our study as well as in previous research (Zheng et al., 2007 2007 ). To solve this problem, we used the C/N-5hmC and C/N-5mC ratios, in which the 5hmC and 5mC levels in cancerous tissues were corrected by those in normal tissues. Global DNA hypomethylation is one of the first epigenetic changes in CRC to be recognized, and we also found lower 5mC levels in different CRC subgroups than in adjacent normal tissues. We further found that decreased C/N-5mC ratios were significantly associated with older cancer patients and those with deeper tumor invasion, which was consistent with the reduction of 5mC levels in cancerous tissues. Moreover, lower 5mC levels were found at early TNM stages than in corresponding normal tissues, suggesting that 5mC plays an important role in the initiation of neoplastic transformation (Suzuki et al., 2006 2006 ). Further reduction of C/N-5mC ratios was found in tumors with infiltrative growth. However, there was no significant correlation between C/N-5mC ratios and TNM stages. One explanation is that DNA hypomethylation occurs in the early stages of tumor development and does not progress with advancing stages, supporting the transient nature of the DNA demethylation process (Haffner et al., 2011 2011 ). Global hypomethylation increases chromosomal instability, while hypermethylation at CpG dinucleotide-dense regions suppresses gene expression, especially the inactivation of tumor suppressor genes. Some studies have also shown that “cancer-germline” genes are activated as a consequence of genome hypomethylation (de Smet and Loriot, 2010 2010 ; 2013 2013 ). Cancer germline genes aberrantly activated in cancer are tissue-specific, and encode tumor-specific antigens. These results revealed a dual effect of DNA hypomethylation in suppression at a later stage of colorectal tumorigenesis, but promotion of early lesions in CRC (Yamada et al., 2005 2005 ). In addition, Kaplan-Meier survival analysis revealed that high C/N-5mC ratios are associated with a better survival outcome and may be an independent prognostic marker. 5hmC, an oxidation product of 5mC, is not only an intermediate in DNA demethylation, but also a stable DNA base involved in gene regulation and tumor genesis (Kudo et al., 2012 2012 ; Pfeifer et al., 2013 2013 ; Uribe-Lewis et al., 2015 2015 ). In this study, we found that in cancerous tissues, 5hmC levels were also lower in different CRC subsets than in adjacent normal tissues. Furthermore, significantly positive correlations between 5mC levels and 5hmC levels were found both in CRC tissues and adjacent normal tissues. Studies of embryonic stem cells have reported that 5hmC may be involved in the regulation of cell differentiation, and its loss in CRC may provide support for the hypothesis that 5hmC plays an important role in cellular differentiation. Since the generation of 5hmC requires 5mC as a substrate, the decreased 5hmC levels may be partly due to reduced 5mC levels (Pfeifer et al., 2013 2013 ). However, in some CRC cases, high levels of 5mC corresponded with low 5hmC levels, suggesting the existence of other mechanisms underlying the reduction in 5hmC. The mRNA levels of the Dnmt (DNA methyltransferase) and Tet families were also assessed, and the correlations with 5hmC levels were analyzed (data not shown). The expression of Dnmt3b was negatively associated with 5hmC levels in tumor tissues, while no significant relationship was found between Tets and 5hmC levels. Other than the Tet family, many enzymes are involved in the process of demethylation, like isocitrate dehydrogenase and thymine DNA glycosylase, which also contributed to the complexity of the results in our study (Kohli and Zhang, 2013 2013 ). Some researchers have reported that Dnmt3a and Dnmt3b not only serve as DNA methyltransferases, but also serve as DNA dehydroxymethylases (Chen et al., 2012 2012 ; Kudo et al., 2012 2012 ). Other studies have also reported that 5hmC can resist DNA hypermethylation (Uribe-Lewis et al., 2015 2015 ). Furthermore, we cannot ignore the possibility that enhanced proliferation leads to a “passive” dilution of 5hmC (Lian et al., 2012 2012 ). Finally, we analyzed the relationships between 5hmC levels and the clinical features of CRC. With regard to the 5hmC ratios, a higher C/N-5hmC value was associated with an advanced TNM stage and the presence of lymph node metastasis. Interestingly, whether in cancerous tissues or in C/N-5hmC ratio, our study showed that a low 5hmC level was associated with a better survival rate using Kaplan-Meier with the P values of 0.04 and 0.08, respectively. However, there was no significant difference with the Cox proportional hazards regression model. The difference might be due to the different sensitivities between the Cox proportional hazards regression model and Kaplan-Meier. High 5hmC levels correlated with poor survival has also been reported in acute myeloid leukemia (Kroeze et al., 2014 2014 ); but studies of other malignancies have shown that 5hmC is a protective factor in the prognosis. Thus, the role of 5hmC in prognosis needs further exploration. We demonstrated that epigenetics plays an important role during tumorigenesis by showing the clinical relevance of 5mC and 5hmC levels in a cohort of CRC patients. Our findings support the hypothesis that loss of 5mC and 5hmC is involved in CRC tumorigenesis. In addition, our results showed that C/N-5mC and C/N-5hmC ratios may be independent prognostic factors for CRC survival outcomes. As far as we know, this is the first study to evaluate the combined effect of C/N-5mC and C/N-5hmC ratios on survival. 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BCR-ABL1-positive microvesicles transform normal hematopoietic transplants through genomic instability: implications for donor cell leukemia . Leukemia , 2014 . 28 ( 8 ): 1666 - 1675 . 24480987 DOI: 10.1038/leu.2014.51 http://doi.org/10.1038/leu.2014.51 . BCR-ABL1-positive microvesicles transform normal hematopoietic transplants through genomic instability: implications for donor cell leukemia We thank Dr. Iain Charles BRUCE (Department of Physiology, School of Medicine, Zhejiang University, Hangzhou, China) for linguistic review of the manuscript. Compliance with ethics guidelines Yi-ping TIAN, Ai-fen LIN, Mei-fu GAN, Hao WANG, Dan YU, Chong LAI, Dan-dan ZHANG, Yi-min ZHU, and Mao-de LAI declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for which identifying information is included in this article. 10.1631/jzus.B1600314 Project supported by the 111 Project (No. B13026), the National High-Tech R&D Program (863) of China (No. 2012AA02A601), the Fundamental Research Funds for the Central Universities, and the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents 2016-07-09 2016-10-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 18