Aberant DNA Methylation In The Promoter Region Of TMEM176A In Esophageal Cancer

Background Esophageal cancer (EC) is one of the most common malignancies in the worldwide and is the sixth leading cause of cancer-related mortality. Esophageal adenocarcinoma (EAC) and squamous cell carcinoma (SCC) are two most common histological types of EC, of whose incidence varies widely by region, with no differences in incidence rates between men and women. EAC predominates in western countries and SCC predominates in Asia. There are an estimated 17040 new cases and 15070 deaths in the United States annually, and it has been reported that the incidence rate of SCC is 18.6% in China. EC occurs at a rate 20 to 30 times higher in our country than in the United States. Esophageal carcinogenesis is a multistage and progressive process which includes basal cell hyperplasia (BCH), dysplasia (DYS), carcinoma in site (CIS) and advanced esophageal carcinoma. A variety of genetic changes and epigenetic modifications participate in the development of EC, especially for the frequent occurrence of DNA hypermethylation of tumor suppressor genes (TSGs). Transmembrane protein 176 (TMEM176) is associated with membrane-spanning 4A family of protein, and TMEM176A is one of the subtypes, which is located at 7q36.1, which shows allelic loss in a variety of tumors. It has been reported that abnormal DNA methylation of TMEM176A is found in breast cancer. However, the expression and function of TMEM176A gene in EC is still unknown.Objective The aim of this Study is to investigate the epigenetic change and the mechanism of TMEM176A in EC.Methods Semi-quantitive RT-PCR (SqRT-PCR) and Methylation specific PCR (MS-PCR) are employed to screen the expression and promoter region methylation of TMEM176A in colon cancer cell lines. Then we analyzed the expression of TMEM176A in EC cell lines to explore if TMEM176A gene is regulated by promoter region methylation. And 103 cases of primary esophageal cancer was employed in this study.Results Frequent mathylation of TMEM176A gene was found in colon cancer, and its expression was regulated by promtor region methylation. Loss of TMEM176A expression and complete methylation of promtor region were detected in TE1、TE3、 TE13、KYSE140、KYSE180、KYSE410、KYSE450、KYSE520、Seg1、YES2、 Colo680 cell lines, high expression of TMEM176A and unmethylation were discovered in BiC1 cell. Restoration of TMEM176A expression or increased expression of TMEM176A was achieved by treating with 5-aza-2’-deoxy-cytidine (5-AZA) in unexpressed and reduced cells. These results demonstrated that the expression of TMEM176A was regulated by DNA methylation of promoter region in EC cell lines. DNA hypermethylation was uncovered in 61.2%(63/103) of primary EC samples. There is no correlation between DNA hypermethylation of TMEM176A and tumor size of EC, tumor stage, tumor differentiation and lymph node metastasis (P>0.05).Conclusion The expression of TMEM176A gene is regulated by DNA methylation in EC cell lines and it is frequently methylated in primary EC. And its biological function and clinical significance need further study.