Hypermethylation of EDNRB promoter contributes to the risk of colorectal cancer

  • Cheng Chen1, 2,

    Affiliated with

    • Lingyan Wang3,

      Affiliated with

      • Qi Liao1,

        Affiliated with

        • Yi Huang4,

          Affiliated with

          • Huadan Ye1,

            Affiliated with

            • Fei Chen1,

              Affiliated with

              • Leiting Xu1,

                Affiliated with

                • Meng Ye2Email author and

                  Affiliated with

                  • Shiwei Duan1Email author

                    Affiliated with

                    Diagnostic Pathology20138:199

                    DOI: 10.1186/1746-1596-8-199

                    Received: 22 October 2013

                    Accepted: 6 December 2013

                    Published: 10 December 2013

                    Abstract

                    Objective

                    Colorectal cancer (CRC) is one of the most common digestive malignancies in the world. EDNRB is a new candidate tumor suppressor gene which is often down-regulated or even silenced by promoter hypermethylation in various human cancers. However, the function of EDNRB gene in CRC remains unknown. In this study, we examined the expression and DNA methylation of EDNRB in CRC tissues.

                    Methods

                    A total of 42 paired CRC and adjacent normal tissue samples were used to determine mRNA levels and DNA methylation status of EDNRB gene by qRT-PCR and methylation-specific PCR (MSP), respectively.

                    Results

                    Our study showed that EDNRB promoter hypermethylation was more frequently in CRC tissues than in the normal tissues (92.86 versus 59.52, p = 0.001). Consequently, significantly lower level of EDNRB mRNA was found in CRC tumor samples than in normal samples (0.31 ± 0.91 versus 0.70 ± 1.18, p = 0.032).

                    Conclusions

                    Our results suggested that EDNRB promoter hypermethylation might downregulate its gene expression in CRC, and thus played an important role in the development of CRC.

                    The virtual slide

                    The virtual slides for this article can be found here: http://​www.​diagnosticpathol​ogy.​diagnomx.​eu/​vs/​7420980471113303​

                    Keywords

                    Colorectal cancer EDNRB DNA methylation Biomarker

                    Introduction

                    Colorectal cancer (CRC) is a malignant disease caused by a variety of factors involving with the accumulation of genetic and epigenetic changes. The incidence of CRC is increasing rapidly, and there are 1 million new CRC cases annually [1]. In the developed countries, the mortality of CRC has soared up to 33. CRC has become the second most common cancer in the world [2], accounting for 9 of all malignant tumors deaths [3]. Although 5-year survival rate of CRC patients has been improved from 22 to 47 in the last 30 years due to the advancement of the early diagnosis, surgical techniques and adjuvant therapies, the overall survival rate remains disappointing.

                    Abberrant expression of some important genes, such as beta-catenin[4], SATB1[5] and EGFR[6], were shown to be significantly associated with the occurrence and prognosis of CRC. Gene promoter hypermethylation often silences gene expression, while promoter hypomethylation tends to activate gene transcription. DNA methylation alteration has been considered as an important event in many malignancies including CRC.

                    Endothelin receptor type B (EDNRB) gene is located on 13q22, encoding a nonselective endothelin B receptor (ETBR) which belongs to a super-family of G-protein coupled receptor that mediates endothelins (ETs) [7]. ETBR is able to promote the production of neural crest cell-specific lineage, and thus it is related to the occurrence of Hirschsprung’s disease [8] that is a blockage in the colon.

                    There are massive CpG dinucleotide repeats in the 5′-flanking region of EDNRB gene. The methylation of this CpG-riched region was shown to be able to regulate EDNRB gene expression [911]. Hypermethylation of EDNRB gene promoter has been observed in leukemia [12], oral cancer [13], skin cancer [14], head and neck cancer [15], melanoma [16], renal cell carcinoma [17], bladder cancer [18] and prostate cancer [19]. However, the function of EDNRB gene in CRC remains unknown. In light of previous findings in various cancers, we investigate whether EDNRB gene promoter methylation contributes to the risk of CRC in Chinese population.

                    Materials and methods

                    Tissue samples collection

                    Tumor and its normal adjacent tissue samples were collected at the time of surgery from 42 primary sporadic CRC patients in the Department of Gastrointestinal Surgery in Affiliated Hospital of Ningbo University between June 2012 and April 2013. Normal adjacent tissues were collected at least 5 cm away from the tumor. Tissues were stored in liquid nitrogen at -80°C immediately after excision. The diagnoses of all CRC cases were pathologically confirmed. None of CRC patients had received preoperative chemotherapy or radiation therapy. Tumor stage was determined according to the Duke’s staging system, and cellular differentiation was graded according to the Broders’ grading system. All the patients in the study have signed the informed written consent forms.

                    DNA isolation

                    Genomic DNA from tissue samples were extracted with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. Briefly, tissue sections were incubated with 180 μl of buffer ATL (in QIAamp DNA Mini Kit) and 20 μl of proteinase K on a thermostatic water bath at 56°C for 3 h, followed by incubation at 70°C for 10 min. Then, 200 μl of remixed Buffer AL (in QIAamp DNA Mini Kit) and ethanol (ratio 1:1) were added. Samples were mixed and transferred into QIAamp Mini spin columns. Centrifugation at 8,000 rpm for 1 min was followed by washing the spin column membrane with 500 μl of Buffer AW1 and 500 μl of Buffer AW2. DNA was eluted with 100 μl of Buffer AE. DNA concentration and quality were determined with spectrophotometer.

                    Bisulfite modification

                    Eluted DNA was bisulphite-treated with ZYMO EZ DNA Methylation-Gold Kit according to the manufacturer’s instruction (Zymo Research, Orange, CA, USA). The bisulphite-modified DNA was resuspended in 10 μl of TE buffer.

                    Methylation-specific PCR (MSP)

                    Methylation status of EDNRB promoter was determined by MSP. For the PCR reaction, 2 μl of modified DNA was amplified in a 20 μl reaction containing 0.3 μM each of forward and reverse primers, 0.2 mM dNTPs, 10 × PCR Buffer and 2.5 U of Hot Start DNA Polymerase (Qiagen, Hilden, Germany) under the following conditions: 15 min of denaturation at 95°C followed by 35 cycles of 45 s at 94°C, 45 s at 62°C for methylated primers, 1 min at 72°C and a final extension for 10 min at 72°C. The sequences of methylated and unmethylated primers were given in the previous study [20] (Table 1). Water blank was used as a negative control. PCR products were subjected to 2.0 agarose gel electrophoresis at 100 V for 20 min, and visualized directly under UV illumination. Samples were considered as methylation or unmethylation, when there were clearly visible bands (130 or 134 bp) on the gel for methylation or unmethylation primers.
                    Table 1

                    List of all primers used and conditions of PCR amplification

                     

                    Primer

                    Sequence (5′-3′)

                    Product size (bp)

                    M

                    F

                    CGAAGAGGTTGCGGGCGGTATTAGCG

                    130

                     

                    R

                    TACTCCAAAAACGTCCGATAACCG

                     

                    U

                    F

                    TGGTGAAGAGGTTGTGGGTGGTATTAGTG

                    134

                     

                    R

                    ACCTACTCCAAAAACATCCAATAACCA

                     

                    RT-PCR

                       
                     

                    EDNRB-F

                    GAAAGCCTCCGTGGGAATC

                    86

                     

                    EDNRB-R

                    ACAGCTCGATATCTGTCAATACTCAGA

                     
                     

                    GAPDH-F

                    CCATGGAGAAGGCTGGGG

                    194

                     

                    GAPDH-R

                    CAAAGTTGTCATGGATGACC

                     

                    RNA isolation and reverse transcription

                    Total RNA was extracted from fresh frozen CRC and normal tissues from the same patients. RNA was extracted with TRIZOL reagent (Invitrogen Life Technologies Co, USA) according to the manufacturer’s protocol. RNA concentration and quality were determined by NanoDrop ND-1000 (Thermal Fisher Scientific, USA). The first-strand cDNA was synthesized according to the manufacturer’s instruction of M-MLV Reverse Transcriptase (Promega, Wisconsin, USA) with 2 μg DNase I-treated total RNA and 2 μM Oligo (dT)15 primer in a 20 μL volume.

                    qRT-PCR

                    Primers for qRT-PCR were listed in Table 1[21]. The qRT-PCR reactions were conducted in a 96-well plate using ABI7500 Real-Time system (Applied Biosystems, CA, USA). Each reaction was performed in triplicate and in a 20 μL volume containing 2× real-time PCR Master Mix with SYBR Green dye (Promega, Wisconsin, USA), 0.4 μM of each primer and 4 μL cDNA, using the following thermal conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min and 72°C for 40 s. A melting curve analysis from 60°C to 95°C was performed at the end of each PCR to further confirm the specificity of amplicons. GAPDH was used as an endogenous control. The Ct values displayed by the instrument were recorded. Samples were confirmed whether there was a clearly visible band (86/194 bp) on the gel with EDNRB or GAPDH primers (Figure 1).
                    http://static-content.springer.com/image/art%3A10.1186%2F1746-1596-8-199/MediaObjects/13000_2013_909_Fig1_HTML.jpg
                    Figure 1

                    EDNRB and GAPDH mRNA expression in CRC tumor tissue.

                    Statistics

                    Statistical analysis was performed by the SPSS statistical package (version 16.0; SPSS, Chicago, IL, USA) and the results were presented using GraphPad prism software. Comparisons of EDNRB promoter methylation were performed by the correction formula of Chi-square test. The 2–ΔCt method was used to analyze the result of qRT-PCR. Two groups of related data were analyzed using paired t-test. The Mann-Whiteney U test was used for two groups of independent data which did not meet normality test. All analyses were two-sided, and p < 0.05 was considered statistically significant.

                    Results

                    Hypermethylated EDNRB promoter in CRC

                    To determine methylation of EDNRB gene, MSP was performed on 42 CRC and 42 adjacent normal tissues in the primary sporadic CRC patients. The representative agarose gel electrophoresis results were shown in Figure 2. Methylation status was determined when there were methylated bands in the gel. And unmethylation status was determined if both the methylated and unmethylated bands were not detected, For the unmethylated samples, we repeated the relative experiment two times, including testing the DNA quality, bisulphite modification and MSP (confirming PCR primers quality and PCR conditions). If the methylated and unmethylated bands still did not detect, we confirmed the status of the sample as unmethylation. Our study showed that hypermethylation of EDNRB in CRC tissues was more frequently than in corresponding normal tissues (92.86 versus 59.52, p = 0.001, Table 2).
                    http://static-content.springer.com/image/art%3A10.1186%2F1746-1596-8-199/MediaObjects/13000_2013_909_Fig2_HTML.jpg
                    Figure 2

                    Representative results for methylation status of EDNRB gene in CRC tumor tissues (T) and adjacent normal tissues (N).

                    Table 2

                    Methylation status of EDNRB gene in CRC and normal tissues

                     

                    Total

                    M

                    U

                    M%

                    χ2

                    p value

                    CRC cases

                    42

                    39

                    3

                    92.86

                    11.091

                    0.001

                    Controls

                    42

                    25

                    17

                    59.52

                      

                    Correlation of EDNRB methylation with clinicopathological characteristics

                    The relationship between methylation status of EDNRB gene and the clinicopathological characteristics of CRC was shown in Table 3. There was no significant difference in clinicopathological factors such as gender, age, TNM stages, lymph node status, metastasis status, tumor location, differentiation status, tumor size, and histological grade. There was no correlation of EDNRB gene methylation status with the serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19-9).
                    Table 3

                    Association between the EDNRB methylation in CRC serum and clinicopathologicalfeatures

                       

                    EDNRB Methylation

                      

                    Characteristics

                     

                    n

                    M

                    U

                    χ2

                    p value

                    Gender

                    Male

                    28

                    27

                    1

                    0.404

                    0.525

                     

                    Female

                    14

                    12

                    2

                      

                    Age(year)

                    ≤60

                    16

                    16

                    0

                    0.629

                    0.428

                     

                    >60

                    26

                    23

                    3

                      

                    Stage

                    1/2

                    21

                    18

                    3

                    1.436

                    0.231

                     

                    3/4

                    21

                    21

                    0

                      

                    Lymph metastasis

                    Yes

                    21

                    21

                    0

                    1.436

                    0.231

                     

                    No

                    21

                    18

                    3

                      

                    Distant metastasis

                    Yes

                    8

                    8

                    0

                    0.012

                    0.913

                     

                    No

                    34

                    31

                    3

                      

                    CEA

                    ≥5.0 ng/ml

                    15

                    14

                    1

                    <0.001

                    1

                     

                    <5.0 ng/ml

                    27

                    25

                    2

                      

                    CA19-9

                    ≥37U/ml

                    9

                    8

                    1

                    <0.001

                    1

                     

                    <37U/ml

                    33

                    31

                    2

                      

                    Tumor location

                    Colon

                    26

                    24

                    2

                    <0.001

                    1

                     

                    Rectum

                    16

                    15

                    1

                      

                    Differentiation

                    Poor

                    10

                    10

                    0

                    0.091

                    0.763

                     

                    Moderate

                    32

                    29

                    3

                      
                     

                    Well

                    0

                    0

                    0

                      

                    Tumor size

                    <5 cm

                    28

                    25

                    3

                    0.404

                    0.525

                     

                    ≥5 cm

                    14

                    14

                    0

                      

                    Histological classification

                    Adenocarcinoma

                    40

                    37

                    3

                    <0.001

                    1

                     

                    Mucinous adenocarcinoma

                    2

                    2

                    0

                      
                     

                    Undifferentiated carcinoma

                    0

                    0

                    0

                      

                    EDNRB expression is down-regulated in CRC tissues

                    We determined EDNRB mRNA expression in 42 CRC tissues and 42 adjacent normal colorectal tissues by qRT-PCR. The gene expression level of EDNRB was normalized with the values of the control gene GAPDH. Our results showed that EDNRB mRNA level in CRC tumor samples was significantly lower than in their adjacent normal samples (0.31 ± 0.91 versus 0.70 ± 1.18, p = 0.032, Figure 3, Table 4).
                    http://static-content.springer.com/image/art%3A10.1186%2F1746-1596-8-199/MediaObjects/13000_2013_909_Fig3_HTML.jpg
                    Figure 3

                    Expression level of EDNRB gene in CRC tumor tissues (T) and adjacent normal tissues (N). RT-qPCR was used to analyze the expression level of EDNRB gene in CRC tumor tissues and adjacent normal tissues. The relative expression of EDNRB mRNA level was significantly higher in normal tissues than that in CRC tumor tissues (p = 0.032).

                    Table 4

                    EDNRB mRNA expression levels in CRC and normal tissues*

                     

                    Total

                    Mean

                    SD

                    SE

                    t

                    p value

                    CRC

                    42

                    0.3074

                    0.91

                    0.14

                    -2.214

                    0.032

                    Control

                    42

                    0.7041

                    1.18

                    0.18

                      

                    *: SD denotes standard deviation and SE denotes standard error.

                    Correlation of EDNRB expression status with clinicopathological characteristics

                    We further examined the relationship between EDNRB mRNA expression and clinicopathological characteristics (Table 5). There was no significant correlation between EDNRB expression and the clinicopathological factors such as gender, age, TNM stages, lymph node status, metastasis status, tumor location, differentiation status, tumor size, and histological classification.
                    Table 5

                    Correlation of EDNRB mRNA expression and clinicopathological parameters of colorectal cancer samples*

                       

                    EDNRB expression relative to GAPDH

                     

                    Characteristics

                     

                    n

                    Mean

                    SD

                    SE

                    p value

                    Gender

                    Male

                    28

                    0.336

                    1.073

                    0.202

                    0.722

                     

                    Female

                    14

                    0.250

                    0.480

                    0.130

                     

                    Age (year)

                    ≤60

                    16

                    0.221

                    0.444

                    0.111

                    0.836

                     

                    >60

                    26

                    0.360

                    1.114

                    0.218

                     

                    Stage

                    1/2

                    21

                    0.143

                    0.233

                    0.051

                    0.792

                     

                    3/4

                    21

                    0.472

                    1.257

                    0.281

                     

                    Lymph metastasis

                    Yes

                    21

                    0.457

                    1.266

                    0.276

                    0.95

                     

                    No

                    21

                    0.158

                    0.238

                    0.052

                     

                    Distant metastasis

                    Yes

                    8

                    0.781

                    1.996

                    0.706

                    0.741

                     

                    No

                    34

                    0.196

                    0.351

                    0.060

                     

                    CEA

                    ≥5.0 ng/ml

                    15

                    0.121

                    0.156

                    0.040

                    0.723

                     

                    <5.0 ng/ml

                    27

                    0.411

                    1.127

                    0.217

                     

                    CA19-9

                    ≥37U/ml

                    9

                    0.102

                    0.153

                    0.051

                    0.526

                     

                    <37U/ml

                    33

                    0.364

                    1.023

                    0.178

                     

                    Tumor location

                    Colon

                    26

                    0.428

                    1.145

                    0.225

                    0.351

                     

                    Rectum

                    16

                    0.112

                    0.156

                    0.039

                     

                    Differentiation

                    Poor

                    10

                    0.719

                    1.779

                    0.558

                    0.494

                     

                    Moderate

                    32

                    0.179

                    0.341

                    0.062

                     
                     

                    Well

                    0

                    /

                    /

                    /

                     

                    Tumor size

                    <5 cm

                    28

                    0.386

                    1.102

                    0.208

                    0.968

                     

                    ≥5 cm

                    14

                    0.151

                    0.248

                    0.066

                     

                    Histological classification

                    Adenocarcinoma

                    40

                    0.315

                    0.935

                    0.148

                    0.455

                     

                    Mucinous adenocarcinoma

                    2

                    0.162

                    0.157

                    0.111

                     
                     

                    Undifferentiated carcinoma

                    0

                    /

                    /

                    /

                     

                    *: SD denotes standard deviation and SE denotes standard error.

                    Discussion

                    Cancer development and progression may be contributed by both genetic and epigenetic factors. As one of the major epigenetic modifications, DNA methylation of promoter often down-regulates gene transcription. A growing number of evidences show that DNA methylation mediated tumor suppressor gene silencing may contribute to tumor progression [2224]. Aberrant DNA methylated loci have become promising biomarkers in the early diagnosis of diseases [25, 26].

                    EDNRB is a G protein–coupled receptor that activates a phosphatidylinositol-calcium second messenger system. The product of EDNRB gene (ETB receptor, ETBR) is able to bind to endothelins (ETs), consisting of a family of 3 potent vasoactive peptides (ET1, ET2, and ET3) [13]. During the development of tumor, EDNRB gene transcription is downregulated by promoter hypermethylated, and consequently alters the ET1 signaling pathway [27]. Disruption in the ET1 signaling pathway has been shown to be involved in a variety of human tumor proliferation, angiogenesis, and metastasis [2830]. EDNRB gene silencing by promoter hypermethylation has been reported in a variety of tumors such as leukemia, oral cancer, skin cancer, head and neck cancer, melanoma, renal cell carcinoma, bladder cancer, and prostate cancer [1219]. Our study in CRC adds a new piece of evidence for the contribution of EDNRB promoter hypermethylation to the risk of CRC. Our results further confirmed that DNA methylation in the promoter region played a key role in EDNRB transcription.

                    Some reports have focused on the correlation between EDNRB methylation and cancer clinical features [3133]. Hypermethylation of the EDNRB gene in paired gastric cancer tissues and adjacent normal tissues from 96 patients was detected [32]. EDNRB gene promoter methylation was shown to be associated with gastric cancer tumor invasion [32]. The extent of hypermethylation at CpG island in EDNRB gene was also evaluated in seven prostate cancer cell lines, normal prostate epithelial cells, normal prostate stromal cells, 73 primary prostate cancers, 91 metastatic prostate cancers, and 25 noncancerous prostate tissues [33]. EDNRB CpG island hypermethylation was shown to be correlated with pathological stage and Gleason score to a statistically significant extent in prostate cancer [33]. In the present study, we examined the relationship between the EDNRB methylation and the clinical features. Unfortunately, there was no significant relationship between EDNRB methylation or expression status and the clinical features. And this may be due to a lack of power in our samples. As a kind of glycoprotein produced by colorectal cancer tissue, CEA is a good tumor marker for judging efficacy, disease progression, and prognosis of colorectal cancer. CA19-9 was frequently increased in gastrointestinal tumors such as pancreatic cancer, gastric cancer, and colorectal cancer. CEA and CA19-9 are commonly and traditionally used in clinical colorectal cancer detection. Our study showed no correlation of EDNRB gene methylation status with the serum levels of CEA and CA19-9. This might imply that aberrant EDNRB methylation and conventional tumor markers could serve as complementary markers in the CRC diagnosis, although further work is needed to confirm our hypothesis.

                    In conclusion, we revealed a significant contribution of EDNRB hypermethylation to the risk of CRC. These findings may provide a new clue for detection and treatment of CRC. Future research is needed to determine the detailed mechanism of EDNRB gene in the risk of CRC.

                    Authors’ information

                    Cheng Chen, Lingyan Wang, Qi Liao: co-first authors of this work.

                    Abbreviations

                    CRC: 

                    Colorectal cancer

                    EDNRB: 

                    Endothelin receptor type B

                    ETAR: 

                    Endothelin A receptor

                    ETBR: 

                    Endothelin B receptor

                    ETs: 

                    Endothelins.

                    Declarations

                    Acknowledgement

                    The research was supported by the grants from the National Natural Science Foundation of China (31100919 and 81371469), Natural Science Foundation of Zhejiang Province (LR13H020003), K. C. Wong Magna Fund in Ningbo University, Ningbo Social Development Research Projects (2012C50032), and Scientific Innovation Team Project of Ningbo (2011B82014).

                    Authors’ Affiliations

                    (1)
                    Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine, Ningbo University
                    (2)
                    The Affiliated Hospital, Ningbo University
                    (3)
                    Bank of Blood Products, Ningbo No.2 Hospital
                    (4)
                    Department of Neurosurgery, Ningbo First Hospital, Ningbo University

                    References

                    1. Migliore L, Migheli F, Spisni R, Coppede F: Genetics, cytogenetics, and epigenetics of colorectal cancer. J Biomed Biotechnol 2011, 2011:792362.PubMed CentralPubMed
                    2. Siegel R, Ward E, Brawley O, Jemal A: Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin 2011,61(4):212–236.PubMedView Article
                    3. Labianca R, Beretta GD, Kildani B, Milesi L, Merlin F, Mosconi S, et al.: Colon cancer. Crit Rev Oncol Hematol 2010,74(2):106–133.PubMedView Article
                    4. Wangefjord S, Brandstedt J, Lindquist KE, Nodin B, Jirstrom K, Eberhard J: Associations of beta-catenin alterations and MSI screening status with expression of key cell cycle regulating proteins and survival from colorectal cancer. Diagn Pathol 2013, 8:10.PubMed CentralPubMedView Article
                    5. Nodin B, Johannesson H, Wangefjord S, O’Connor DP, Lindquist KE, Uhlen M, et al.: Molecular correlates and prognostic significance of SATB1 expression in colorectal cancer. Diagn Pathol 2012, 7:115.PubMed CentralPubMedView Article
                    6. Dekanic A, Dintinjan RD, Budisavljevic I, Pecanic S, Butorac MZ, Jonjic N: Strong nuclear EGFR expression in colorectal carcinomas is associated with cyclin-D1 but not with gene EGFR amplification. Diagn Pathol 2011, 6:108.PubMed CentralPubMedView Article
                    7. Jojima T, Suzuki K, Hirama N, Uchida K, Hattori Y: Glimepiride upregulates eNOS activity and inhibits cytokine-induced NF-kappaB activation through a phosphoinoside 3-kinase-Akt-dependent pathway. Diabetes Obes Metab 2009,11(2):143–149.PubMedView Article
                    8. Puffenberger EG, Hosoda K, Washington SS, Nakao K, deWit D, Yanagisawa M, et al.: A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell 1994,79(7):1257–1266.PubMedView Article
                    9. Eberle J, Weitmann S, Thieck O, Pech H, Paul M, Orfanos CE: Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes. J Invest Dermatol 1999,112(6):925–932.PubMedView Article
                    10. Pao MM, Tsutsumi M, Liang G, Uzvolgyi E, Gonzales FA, Jones PA: The endothelin receptor B (EDNRB) promoter displays heterogeneous, site specific methylation patterns in normal and tumor cells. Hum Mol Genet 2001,10(9):903–910.PubMedView Article
                    11. Yang AS, Doshi KD, Choi SW, Mason JB, Mannari RK, Gharybian V, et al.: DNA methylation changes after 5-aza-2′-deoxycytidine therapy in patients with leukemia. Cancer Res 2006,66(10):5495–5503.PubMedView Article
                    12. Hsiao PC, Liu MC, Chen LM, Tsai CY, Wang YT, Chen J, et al.: Promoter methylation of p16 and EDNRB gene in leukemia patients in Taiwan. Chin J Physiol 2008,51(1):27–31.PubMed
                    13. Schussel J, Zhou XC, Zhang Z, Pattani K, Bermudez F, Jean-Charles G, et al.: EDNRB and DCC salivary rinse hypermethylation has a similar performance as expert clinical examination in discrimination of oral cancer/dysplasia versus benign lesions. Clin Cancer Res 2013,19(12):3268–3275.PubMedView Article
                    14. Zhang M, Song F, Liang L, Nan H, Zhang J, Liu H, et al.: Genome-wide association studies identify several new loci associated with pigmentation traits and skin cancer risk in European Americans. Hum Mol Genet 2013,22(14):2948–2959.PubMedView Article
                    15. Roh JL, Wang XV, Manola J, Sidransky D, Forastiere AA, Koch WM: Clinical correlates of promoter hypermethylation of four target genes in head and neck cancer: a cooperative group correlative study. Clin Cancer Res 2013,19(9):2528–2540.PubMedView Article
                    16. Cruz-Munoz W, Jaramillo ML, Man S, Xu P, Banville M, Collins C, et al.: Roles for endothelin receptor B and BCL2A1 in spontaneous CNS metastasis of melanoma. Cancer Res 2012,72(19):4909–4919.PubMedView Article
                    17. Wuttig D, Zastrow S, Fussel S, Toma MI, Meinhardt M, Kalman K, et al.: CD31, EDNRB and TSPAN7 are promising prognostic markers in clear-cell renal cell carcinoma revealed by genome-wide expression analyses of primary tumors and metastases. Int J Cancer 2012,131(5):E693-E704.PubMedView Article
                    18. Zuiverloon TC, Beukers W, van der Keur KA, Munoz JR, Bangma CH, Lingsma HF, et al.: A methylation assay for the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences in voided urine. BJU Int 2012,109(6):941–948.PubMedView Article
                    19. Vasiljevic N, Wu K, Brentnall AR, Kim DC, Thorat MA, Kudahetti SC, et al.: Absolute quantitation of DNA methylation of 28 candidate genes in prostate cancer using pyrosequencing. Dis Markers 2011,30(4):151–161.PubMedView Article
                    20. de Freitas C-SM, Oliveira ZF, de Podesta JR, Gouvea SA, Von Zeidler SV, Louro ID: Methylation analysis of cancer-related genes in non-neoplastic cells from patients with oral squamous cell carcinoma. Mol Biol Rep 2011,38(8):5435–5441.View Article
                    21. Tang W, Li B, Tang J, Liu K, Qin J, Wu W, et al.: Methylation analysis of EDNRB in human colon tissues of Hirschsprung’s disease. Pediatr Surg Int 2013,29(7):683–688.PubMedView Article
                    22. Shen WJ, Dai DQ, Teng Y, Liu HB: Regulation of demethylation and re-expression of RASSF1A gene in gastric cancer cell lines by combined treatment of 5-Aza-CdR and NaB. World J Gastroenterol 2008,14(4):595–600.PubMedView Article
                    23. Du YY, Dai DQ, Yang Z: Role of RECK methylation in gastric cancer and its clinical significance. World J Gastroenterol 2010,16(7):904–908.PubMed
                    24. Rizvi MM, Alam MS, Ali A, Mehdi SJ, Batra S, Mandal AK: Aberrant promoter methylation and inactivation of PTEN gene in cervical carcinoma from Indian population. J Cancer Res Clin Oncol 2011,137(8):1255–1262.PubMedView Article
                    25. Kang GH, Lee S, Kim JS, Jung HY: Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis. Lab Invest 2003,83(5):635–641.PubMedView Article
                    26. Tost J: DNA methylation: an introduction to the biology and the disease-associated changes of a promising biomarker. Mol Biotechnol 2010,44(1):71–81.PubMedView Article
                    27. Davenport AP, Maguire JJ: Endothelin. Handb Exp Pharmacol 2006,176(1):295–329.PubMedView Article
                    28. Zhao BJ, Sun DG, Zhang M, Tan SN, Ma X: Identification of aberrant promoter methylation of EDNRB gene in esophageal squamous cell carcinoma. Dis Esophagus 2009,22(1):55–61.PubMedView Article
                    29. Lahav R, Heffner G, Patterson PH: An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells in vitro and in vivo. Proc Natl Acad Sci USA 1999,96(20):11496–11500.PubMedView Article
                    30. Mallat A, Fouassier L, Preaux AM, Gal CS, Raufaste D, Rosenbaum J, et al.: Growth inhibitory properties of endothelin-1 in human hepatic myofibroblastic Ito cells. An endothelin B receptor-mediated pathway. J Clin Invest 1995,96(1):42–49.PubMed CentralPubMedView Article
                    31. Nelson J, Bagnato A, Battistini B, Nisen P: The endothelin axis: emerging role in cancer. Nat Rev Cancer 2003,3(2):110–116.PubMedView Article
                    32. Tao K, Wu C, Wu K, Li W, Han G, Shuai X, et al.: Quantitative analysis of promoter methylation of the EDNRB gene in gastric cancer. Med Oncol 2012,29(1):107–112.PubMedView Article
                    33. Yegnasubramanian S, Kowalski J, Gonzalgo ML, Zahurak M, Piantadosi S, Walsh PC, et al.: Hypermethylation of CpG islands in primary and metastatic human prostate cancer. Cancer Res 2004,64(6):1975–1986.PubMedView Article

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