Methylation Quantification of Tumor Suppressor Genes in NPC by Methylation-Sensitive High Resolution Melting Analysis

• Main Authors: Shao Tung Huang, 黃少東
• Other Authors: P. H. Li
• Format: Others
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/89909914537481784618

碩士 === 長庚大學 === 生物醫學研究所 === 100 === Aberrant DNA hypermethylation of tumor suppressor genes (TSGs) inhibits gene expression and causes tumor progression. Since TSGs hypermethylation often used as biomarkers reflecting tumor progression, we used methylation specific high resolution melting (MS-HRM) PCR-based analysis to rapidly detect DNA methyation percentage of aberrant hypermethylated genes in nasopharyngeal carcinoma (NPC). We previously identified a novel hypermethylated gene, Gene A, in NPC biopsies. Gene A is a transcription factor which regulates body axes development. According to our bisulfite sequencing data , Gene A was hypermethylated in NPC cell lines and NPC tumor biopsies (65~95%), however, Gene A gene of adjacent normal tissues and normal individuals’ white blood cells was hypomethylated (8~65%). To test whether MS-HRM has comparable results as bisulfite sequencing, we performed MS-HRM using the same samples. Indeed, MS-HRM had consistent results as bisulfite sequencing. Besides, MS-HRM is a more rapid and economic analysis when compared with bisulfite sequencing . Since EBV copy number is considered as prognostic marker for NPC, we isolated 27 cell-free DNA samples from 5 NPC patients’ plasma at difference time. We further detected EBV copy number and Gene A methylation, respectively. Our results indicated that EBV copy number was positively correlated with Gene A methylation . Hence, the copy number of EBV in plasma could reflect Gene A methylation level in tumor. Taken together, we have developed a rapid and accurate MS-HRM method to detect Gene A hypermethylation; we may use this gene as a biomarker for NPC.