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

Rawłuszko-Wieczorek AA, Siera A, Horbacka K, Horst N, Krokowicz P, Jagodziński PP - J. Cancer Res. Clin. Oncol. (2015)

Bottom Line: We found reduced transcript levels of TET1, TET2 and TET3 in cancerous tissue compared with their histopathologically unchanged counterparts (p = 0.000011; p = 0.000001; p = 0.00031, respectively).Moreover, we found no DNA methylation in the TET2 and TET3 promoter regions in cancerous and histopathologically unchanged tissue.Additionally, our findings initially indicate the importance of DNA methylation in regulating TET1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences, Poznan, Poland, arawluszko@ump.edu.pl.

ABSTRACT

Purpose: Ten eleven translocation (TET) enzyme activity is essential for active DNA demethylation in biological processes, and their altered expression has been observed in various malignancies. Therefore, we investigated DNA methylation and mRNA levels of all TETs in colorectal cancer (CRC) patients.

Methods: TET mRNA levels were evaluated using quantitative RT-PCR in primary cancerous and histopathologically unchanged colorectal tissues from patients who underwent radical surgical colon resection (n = 113). DNA methylation levels of the TET CpG island were assessed using bisulfite DNA sequencing and high-resolution melting analysis.

Results: We found reduced transcript levels of TET1, TET2 and TET3 in cancerous tissue compared with their histopathologically unchanged counterparts (p = 0.000011; p = 0.000001; p = 0.00031, respectively). Importantly, multivariate Cox regression analysis revealed favorable overall survival (OS) and disease-free survival (DFS) outcomes for patients with high TET2 mRNA levels in histopathologically unchanged tissue (HR(OS) = 0.091, 95 % CI 0.011-0.77, p = 0.028; HR(DFS) = 0.21, 95 % CI 0.04-1.06, p = 0.059). Moreover, we found no DNA methylation in the TET2 and TET3 promoter regions in cancerous and histopathologically unchanged tissue. In contrast, we reported TET1 DNA hypermethylation in a small fraction of patients (n = 12/113).

Conclusion: To best of our knowledge, our study is the first to investigate TET mRNA levels in a cohort of CRC patients and correlate them with patients' prognosis. Present study provides the evidence that TET2 mRNA expression may be an independent prognostic factor for disease recurrence and outcome. Additionally, our findings initially indicate the importance of DNA methylation in regulating TET1 expression.

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DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator
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Fig4: DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator

Mentions: 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).Fig. 2


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

Rawłuszko-Wieczorek AA, Siera A, Horbacka K, Horst N, Krokowicz P, Jagodziński PP - J. Cancer Res. Clin. Oncol. (2015)

DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4469774&req=5

Fig4: DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator
Mentions: 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).Fig. 2

Bottom Line: We found reduced transcript levels of TET1, TET2 and TET3 in cancerous tissue compared with their histopathologically unchanged counterparts (p = 0.000011; p = 0.000001; p = 0.00031, respectively).Moreover, we found no DNA methylation in the TET2 and TET3 promoter regions in cancerous and histopathologically unchanged tissue.Additionally, our findings initially indicate the importance of DNA methylation in regulating TET1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences, Poznan, Poland, arawluszko@ump.edu.pl.

ABSTRACT

Purpose: Ten eleven translocation (TET) enzyme activity is essential for active DNA demethylation in biological processes, and their altered expression has been observed in various malignancies. Therefore, we investigated DNA methylation and mRNA levels of all TETs in colorectal cancer (CRC) patients.

Methods: TET mRNA levels were evaluated using quantitative RT-PCR in primary cancerous and histopathologically unchanged colorectal tissues from patients who underwent radical surgical colon resection (n = 113). DNA methylation levels of the TET CpG island were assessed using bisulfite DNA sequencing and high-resolution melting analysis.

Results: We found reduced transcript levels of TET1, TET2 and TET3 in cancerous tissue compared with their histopathologically unchanged counterparts (p = 0.000011; p = 0.000001; p = 0.00031, respectively). Importantly, multivariate Cox regression analysis revealed favorable overall survival (OS) and disease-free survival (DFS) outcomes for patients with high TET2 mRNA levels in histopathologically unchanged tissue (HR(OS) = 0.091, 95 % CI 0.011-0.77, p = 0.028; HR(DFS) = 0.21, 95 % CI 0.04-1.06, p = 0.059). Moreover, we found no DNA methylation in the TET2 and TET3 promoter regions in cancerous and histopathologically unchanged tissue. In contrast, we reported TET1 DNA hypermethylation in a small fraction of patients (n = 12/113).

Conclusion: To best of our knowledge, our study is the first to investigate TET mRNA levels in a cohort of CRC patients and correlate them with patients' prognosis. Present study provides the evidence that TET2 mRNA expression may be an independent prognostic factor for disease recurrence and outcome. Additionally, our findings initially indicate the importance of DNA methylation in regulating TET1 expression.

Show MeSH
Related in: MedlinePlus