A Preliminary Research On Frequent Methylation Of The CASR Promoter In Human Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer is one of the most common digestive tract tumors in the worldwide. In China, most of cases are esophageal squamous cell carcinoma, which is a highly malignant disease with poor prognosis and seriously threats to human health. However, esophageal cancer patients have no typical symptoms in the early stage, majority of patients diagnosed clinically have missed the best treatment period. Therefore, a key of improvement of the prognosis and life quality for patients is screening of molecular biomarkers for the early diagnosis of esophageal cancer. Calcium-sensing receptor (CASR), a member of the G protein-coupled receptor family, plays an essential role in maintaining mineral ion homeostasis. It has been found that the sequence of CASR promoter is hypermethylation and plays an important role as a potential tumor suppressor in colorectal carcinogenesis. However, there is no similar report in esophageal cancer.ObjectiveThe aim of this research project was to investigate the promoter methylation status and the expression of CASR in esophageal from the angle of epigenetics. Meanwhile, we analyzed methylation condition of CASR promoter in normal esophageal mucosa and ESSC tissues to explore whether the CASR promoter methylation would be used as a potential biomarker for the early diagnosis of human ESCC.MethodsThe promoter methylation status and rates of CASR in6esophageal cancer cell lines were investigated using methylation specific PCR (MSP) and Bisulfite sequencing (BSSQ). Semi-quantitative reverse transcriptase PCR (RT-PCR) and Western Blot (WB) were used to evaluate CASR expression levels before and after treatment with5-aza-2’-deoxycytidine (5-Aza) in the cell lines. So we can clarify whether the expression of CASR is regulated by its promoter methylation status. In the meantime, MSP was employed to detect the promoter methylation status of CASR in8normal esophageal mucosa and68cancer tissue samples from primary ESCC patients and combined with the related clinical datas to further analyze the possible role of CASR in the progress of ESCC by Chi-square test.Results1. The methylation status and rates of CASR promoter in esophageal squamous cell cancer cell lines:We found that the promoter of CASR was completely methylated in KYSE30, KYSE140and BIC1, the methylation rates92.7%,93.6%,87.3%, respectively. Partial methylation of the CASR promoter region was detected in2ESCC cell lines KYSE70and TE8, but not in KYSE510.2. The expression status of CASR in the ESCC cell lines before and after treatment with5-Aza:loss or down-regulation of CASR experssion was found in the methylated cell lines KYSE30, KYSE140and TE8. Re-expression of CASR was induced after5-Aza treatment for96h in cell lines, but not in non-methylated KYSE510.3. The association with CASR methylation status of primary ESCC tissue samples and normal esophageal mucosa:CASR methylation was detected in70.6%(48/68) of human primary ESCC tissue samples but not in normal esophageal mucosa (0/8). No association was found between promoter region hypermethylation and age, gender, tumor location, tumor stage and lymph node metastasis. Conclusions1. The CASR gene is expressed under the control of the methylated sequence of CASR promoter, and its abnormal epigenetic silence may be involved in carcinogenesis of the esophagus.2. The sequence of CASR is frequently methylated in the esophageal cancer cell lines and cancer tissues from ESCC patients, suggesting that the methylation status of CASR promoter may be used as a potential early detection biomarker or therapeutic target for the disease.