Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer

Primary colonic adenocarcinoma tissues were collected between June 2009 and March 2013 from a cohort of 113 patients who underwent radical surgical resection of the colon at the Department of General and Colorectal Surgery, Poznań University of Medical Sciences, Poland (Table 1). Histopathologically unchanged colonic mucosa located at least 10–20 cm away from the cancerous lesions were obtained from the same patients. One set of samples was immediately snap-frozen in liquid nitrogen and stored at −80 °C until DNA and RNA isolation. The other set of samples was directed for histopathological examination performed by an experienced pathologist. None of the patients received preoperative chemo- or radiotherapy. Informed consent was obtained from all participating individuals. The procedures of the study were approved by the Local Ethical Committee of Poznań University of Medical Sciences. Written consent was obtained from all participants.

Follow-up data were available for 88 patients, who were observed from 2009/08/01 until death or 2014/25/05, whichever came first. Disease-free survival (DFS) is defined as the time elapsed from surgery to the first occurrence of any of the following events: recurrence or distant metastasis of CRC or the development of a second non-colorectal malignancy. The diagnosis of recurrence and metastasis was evaluated with a variety of methods, including computed tomography, ultrasonography, position emission tomography, cytologic analysis or biopsy. Overall survival (OS) is defined as the time elapsed from surgery to the death of the CRC patient. Death of patients was verified by public records and family reports. The DFS and OS statuses were ascertained by a physician blinded to the TET mRNA levels.

Genomic DNA from the tissues of patients with CRC was isolated using the DNA Mammalian Genomic Purification Kit purchased from Sigma-Aldrich Co. (St. Louis, MO). Then, 500 ng of genomic DNA was subjected to bisulfite conversion of cytosine to uracil, according to the EZ DNA Methylation Kit™ procedure from Zymo Research Corporation (Orange, CA). The positions of the CpG islands and transcription factor-binding sites located in the TET1, TET2 and TET3 promoters were determined by online programs (CpGPlot/CpGReport/Isochore; Searcher; Site).

The DNA fragments containing CpG dinucleotides located in the promoter region of the TET1, TET2 and TET3 genes were amplified by the primer pairs complementary to the bisulfite-DNA modified sequences (Supplementary Table 1). PCR amplification was performed by FastStart Taq DNA Polymerase from Roche Diagnostic GmbH (Mannheim, Germany). The PCR products were purified using Agarose Gel DNA Extraction Kit, Roche Diagnostic GmbH (Mannheim, Germany) with subsequent cloning into pGEM-T Easy Vector System I, Promega (Madison, WI) and transformation into TOPO10 E. coli strain cells. The plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing of the cloned fragment of DNA. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and bisulfite sequencing data presentation and compilation (BDPC) web server, respectively (Bock et al. 2005; Rohde et al. 2008).

The methylation levels of DNA fragments located within the CpG islands of the TET1, TET2 and TET3 genes were determined by real-time PCR amplification of bisulfite-treated DNA, followed by HRM profile analysis by Light Cycler^®480 or LightCycler^®96 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). For PCR amplification, 1 μl of either the bisulfite-treated DNA from patients or standards, together with primers (Supplementary Table 1), was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia). Standardized solutions with a given DNA methylation percentage were prepared by mixing methylated and non-methylated bisulfite-treated DNA from the Human Methylated/Non-methylated DNA Set, Zymo Research Corp. (Orange, CA) in different ratios. To determine the percentage of methylation, the HRM profiles of patients’ DNA PCR products were compared with HRM profiles of standard DNA PCR products (Rawluszko et al. 2011; Wojdacz and Dobrovic 2007). HRM methylation analysis was performed using the Light Cycler^®480 or LightCycler^®96 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate. The methylation status for each patient was presented as a percentage of methylation in amplified fragments located in the CpG islands of TET1, TET2 and TET3.

Total RNA from the tissues of patients with CRC was isolated according to the method of Chomczyński and Sacchi (1987). The RNA samples were quantified and reverse-transcribed into cDNA. RQ-PCR was carried out in the Light Cycler^®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany) using SYBR^® Green as the detection dye. The target cDNA was quantified by the relative quantification method using a calibrator for the primary tissues. The calibrator was prepared as a cDNA mix from all of the patients’ samples, and successive dilutions were used to create a standard curve as described in Relative Quantification Manual, Roche Diagnostics GmbH (Mannheim, Germany). For amplification, 1 μl of (total 20 μl) cDNA solution was added to 9 μl of IQ™ SYBR^® Green Supermix, Bio-Rad Laboratories Inc. (Hercules, CA) with primers (Supplementary Table 1). To prevent the amplification of sequences from genomic DNA contamination, primers and/or amplicons were designed at exon/exon boundaries and covered all gene splice variants. The quantity of TET1, TET2 and TET3 transcripts in each sample was standardized by the geometric mean of two internal controls: porphobilinogen deaminase (PBGD) and human mitochondrial ribosomal protein L19 (hMRPL19) (Supplementary Table 1). The selection of internal control genes was performed as previously described (Rawluszko-Wieczorek et al. 2014). The TET1, TET2 and TET3 transcript levels in the patients’ tissues were expressed as a multiplicity of the cDNA concentrations in the calibrator. A few samples, in which the quantity of isolated RNA was insufficient to prepare high-quality RQ-PCR products, were excluded from further analysis. Hence, the number of patients in the subgroups of survival analysis may vary by ±4 samples.

The normality of the observed patient data distribution was assessed using the Shapiro–Wilk test, and the median values were compared using the U Mann–Whitney test. Survival curves were plotted using the Kaplan–Meier method, and survival differences were determined using the log rank test. The multivariable Cox proportional hazard model was used to estimate the adjusted hazard ratio (HR). Statistical analysis was performed with the STATISTICA 10.0 software, and p < 0.05 was considered statistically significant.

We used RQ-PCR to compare TET1, TET2 and TET3 transcript levels in cancerous and histopathologically unchanged tissues in 113 patients with CRC. We found significantly lower levels of TET1, TET2 and TET3 mRNA levels (p = 0.000011; p = 0.000001; p = 0.00031, respectively) in cancerous tissues than in histopathologically unchanged tissues (Fig. 1). The lower levels of TET1 and TET2 mRNA levels in cancerous tissues were observed across different age groups, genders, CRC localization, histological grades and TNM classification (Table 2A, B). Differences in the IIC and IIIA TNM classes did not reach statistically significant results for all the TETs, most likely due to the small number of patients in these subgroups. However, age and localization-related expression was observed for the TET3 transcript. Significantly lower TET3 mRNA levels in the cancerous tissues was observed in the groups of patients above 60 years of age, and in tumors localized in the proximal colon (Table 2C).

To compare DNA methylation levels in the TET1, TET2 and TET3 promoter regions between DNA samples from the cancerous and histopathologically unchanged tissues, we performed bisulfite DNA sequencing followed by HRM analysis. Bisulfite sequencing was used for evaluation of DNA methylation in large regions of CpG islands in randomly selected patients. We detected a similar pattern of DNA methylation within all individual clones of each patient. We undetected DNA methylation of TET2 (chr4: 106,067,501–106,068,077) and TET3 (chr2: 74,211,418–74,211,829) promoter regions using bisulfite sequencing in selected patients (Fig. 2b, c). In keeping with bisulfite sequencing data, we have not observed DNA methylation within TET2 (chr4: 106 067,537–106,067 735) and TET3 (ch42: 74,211,418–74,211 584) promoter regions in 113 patients with CRC (Fig. 3b, c). Although HRM analysis revealed no DNA methylation in TET1 promoter region (chr10: 70 320 271–70,320 457) of 101 samples, significant DNA hypermethylation in cancerous tissues, compared to histopathologically unchanged tissues, was observed in 12 patients (Fig. 3a). The DNA hypermethylation of TET1-selected region was also observed using bisulfite sequencing (Fig. 2a). Patients numbered P2–P4 are among group with detected DNA methylation in cancerous tissue using HRM analysis (Fig. 2a). Stratification of the patients with increased DNA methylation in the TET1 promoter by gender, age, localization, histological grade and TNM did not reveal any significant tendency (Table 3). Nonetheless, patients with DNA hypermethylation in the TET1 regulatory region of cancerous tissues have lower TET1 transcript levels (p = 0.074), compared to cancerous tissues with no DNA methylation changes in the same region (Fig. 4).

To assess the effect of mRNA and DNA methylation levels of TET gene family members, we performed a retrospective analysis of 88 patients. The patients’ median survival was 38 (range 4–59) months. The transcript levels of TET1, TET2 and TET3 were divided into three groups: low, intermediate and high mRNA levels. The analysis of TET1 transcript levels in histopathologically unchanged tissues and TET2 in cancerous tissue did not reveal statistically significant results (Fig. 5a, c). However, compared to those with intermediate and high TET1 transcript levels, patients with low TET1 mRNA levels in cancerous tissues trended toward favorable DFS outcomes (Fig. 5a). Although these results did not reach statistical significance, low TET1 mRNA levels in cancerous tissue also correlated with increased patient OS, with a median survival of 41 months versus 36 and 33 months for intermediate and high TET1 mRNA levels, respectively (Fig. 5a). Moreover, follow-up data were available for eight out of 12 patients with DNA hypermethylation within CpG islands of the TET1 gene in cancerous tissues. Log rank test analysis did not reveal statistically significant results for the patients with DNA methylation (Fig. 5b). Further, Kaplan–Meier analysis revealed that the CRC patients with high TET2 mRNA level in histopathologically unchanged tissues had better overall and DFS outcomes (OS median: 41 months; DFS median: 40 months) than those with low TET2 mRNA levels (OS median: 26.5 months; DFS median: 28 months) (Fig. 5c). In the present study, analysis of TET3 mRNA levels in histopathologically unchanged and cancerous colorectal tissues revealed no significant correlation with the patients’ OS and DFS (Fig. 5d).

We performed multivariable analysis for TET2 mRNA levels in histopathologically unchanged tissues and for TET1 mRNA level in cancerous tissues. In Cox proportional hazard regression analysis, the categorical variables introduced included age, gender, tumor localization and postoperative chemotherapy. In the current study, adjusted analysis did not reveal statistically significant results for TET1 mRNA levels in cancerous tissues (Table 4). However, multivariable analysis revealed that high TET2 mRNA levels in histopathologically unchanged tissues can be an independent prognostic factor for OS and DFS (Table 5).