Identification of aberrant promoter methylation of EDNRB gene in esophageal squamous cell carcinoma.

Epigenetic silencing of tumor suppressor genes is a major contributor to neoplastic transformation and is an area of intense research. The purpose of the present study was to identify the epigenetic changes in esophageal squamous cell carcinoma (ESCC). Methylation-sensitive arbitrarily primed polymerase chain reaction analysis was used on 21 matched ESCC tumors and adjacent normal tissues. Through this screen we identified a frequently methylated fragment that showed a high homology to the 5' CpG island of endothelin receptor type B (EDNRB) gene. The methylation status of the EDNRB gene was then detected by bisulfite sequencing and the levels of EDNRB mRNA were detected by quantitative real-time polymerase chain reaction (PCR). In addition, the effects of a methylation inhibitor 5-aza-2'-deoxycytidine on EDNRB mRNA expression was determined in cells of an ESCC cell lines. Hypermethylation of the 5' CpG island of EDNRB was found in 5 out of 21 (23.8%) primary tumors. Real-time PCR analysis demonstrated that EDNRB mRNA expression was significantly reduced in tumors showing high promoter methylation compared with paired normal tissues, whereas there is no significant difference between other paired samples. In addition, treatment of ESCC cell line with 5-aza-2'-deoxycytidine led to reexpression of the EDNRB transcript, which is correlated with the reversal of the methylation status of EDNRB promoter. In conclusion, promoter hypermethylation of EDNRB gene, which is associated with the loss of EDNRB mRNA expression, may play a role in the development of ESCC.