Hypermethylation of HoxA2 in Nasopharyngeal carcinoma

• Main Authors: Chen Ching Peng, 彭宸璟
• Other Authors: H. P. Li
• Format: Others
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/73878626579976312823

碩士 === 長庚大學 === 生物醫學研究所 === 98 === DNA methyltransferase 1 (DNMT1), an enzyme which regulates DNA promoter methylation leading to gene silencing, is activated in nasopharyngeal carcinoma (NPC). Aberrant DNA methylation of tumor suppressor gene promoters has been proven to increase the risk of cancers. Thus, DNA methylation may play an important role in NPC tumor formation. In this study, we identified a differential hypermethylated gene HoxA2 in NPC tumor tissues compared with adjacent normal tissues. Next, we performed bisulfite sequencing to examine HoxA2 promoter methylation status. Our data showed that HoxA2 is hypermethylated in -443~ +83 region in NPC tumor tissues. In NPC cell lines, HoxA2 mRNA expression level was restored with the treatment of DNA methylation inhibitor, 5’Aza. In vitro methylated HoxA2 promoter reporter (pHoxA2-Luc) had reduced luciferase activity indicating that methylation can inhibit promoter activity. According to prediction, two transcriptional activator p300 binding sites were found to locate on HoxA2 promoter. Overexpressed p300 whose binding sequence covers 2~ 3 CpG sites could activate HoxA2 promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that the p300 binding affinity on HoxA2 proximal promoter was increased with 5’Aza treatment. These data implied that methylation on HoxA2 promoter may prevent p300 (-136~ -123) binding; therefore, HoxA2 hypermethylation is correlated with gene silencing. We utilized lentivirus expression system to investigate the function of HoxA2. In NPC cell lines, lentivirus overexpressed HoxA2 stable clone had lower cell invasion ability compared with vector control. In addition, gelatin zymography assay demonstrated that lower cell invasion ability correlated with reduction in MMP-9 enzymatic activity and mRNA expression level suggesting that HoxA2 may inhibit cell invasion activity by reducing MMP-9 expression. Overall, these data demonstrated that the expression of HoxA2 is repressed in NPC via DNA methylation and that HoxA2 gene silencing may promote NPC cell invasion ability. Thus, HoxA2 may function as a tumor suppressor gene.