A study on the hypermethylation of tumor suppressor genes in nasopharyngeal carcinoma

Abstract

Studies have shown that promoter hypermethylation of tumor suppressor genes may serve as a promising epigenetic biomarker for the diagnosis of nasopharyngeal carcinoma (NPC), which is of great significance in improving patient's survival rate. Resulting from DNA leakage due to tumor necrosis or apoptosis, cell-free circulating DNA in blood has been proven sharing a similar hypermethylation status as primary tumor and is considered desirable for promoter hypermethylation status screening. This study investigated the potential of promoter hypermethylation of five tumor suppressor genes in the detection of NPC in serum samples. Cell-free circulating DNA was extracted from serum collected from 40 NPC patients before treatment and 41 age- and sex-matched healthy subjects. Promoter hypermethylation status of five tumor suppressor genes (RASSF1A, CDKN2A, DLEC1, DAPK and UCHL1) was assessed by methylation-specific polymerase chain reaction (MSP) after sodium bisulfite conversion. Methylation status of five tumor suppressor genes and clinicopathological parameters (age, gender and staging) were compared between NPC patients and healthy subjects. The concentration of cell-free circulating DNA in the serum of NPC patients was significantly higher than that in normal controls. The five tumor suppressor genes RASSF1A, CDKN2A, DLEC1, DAPK and UCHL1 were found to be methylated in 17.5%, 22.5%, 25%, 51.4% and 64.9% of the patients, respectively. The combination of four-gene markers - CDKN2A, DLEC1, DAPK and UCHL1 -had the highest sensitivity and specificity in predicting NPC. Our results suggested that screening of DNA hypermethylation of tumor suppressor genes in serum may be a promising approach for the diagnosis of nasopharyngeal carcinoma. Promoter hypermethylation has been reported to be associated with the transcription inhibition of tumor suppressor genes and the initiation and progression of tumor cells. To explore the role of three candidate tumor suppressor genes DLEC1, DAPK and UCHL1 in NPC tumorigenesis, changes in methylation status and transcription of these three genes were examined by methylation-specific PCR and RT-PCR in NPC cell line HNE1 treated with the demethylation reagent, 5-aza-2' deoxycytidine. Results showed that promoter hypermethylation could lead to transcriptional silencing of DLEC1, DAPK and UCHL1 in NPC cell line HNE1.