Identification Of Hypermethylated Genes In Nasopharyngeal Carcinoma Cell Line By Proteomics

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China. However, the mechanism underlying the pathogenesis of NPC remains unclear. DNA methylation is one of the epigenetics mechanisms of gene expression regulation. And promoter methylation of tumor suppression genes plays a critical role in carcinogenesis. Therefore, screening methylation silenced genes may help to reveal the pathogenesis of NPC, which has important theoretical and clinical value.Proteomic analysis was performed to screen for methylation silenced genes in NPC cell line 5-8F with high metastatic potential. MTT assay and flow cytometry were used to determine the optimum concentration of demethylating agent 5-aza-2-dC, which was used to treat 5-8F cells. Two-dimensional gel electrophoresis (2-DE) was performed to separate the proteins of treated and untreated 5-8F cells with 5-aza-2-dC. PDQuest software was used to analyze 2-DE images to determine the differential expression proteins between the treated and untreated 5-8F cells. MALDI-TOF-MS was used to identify the differentially expressed proteins. Western blot and RT-PCR analysis were used to examine the expression levels of the differential expression protein nm23-H1. Methylation-specific PCR (MSP) was performed to examine the methylation status of nm23-H1 gene in 5-8F cells. The results were as following: (1) 2-DE reference patterns of 5-8F cells treated and untreated with 5-aza-2-dC were established. Forty-nine differential protein spots were found between treated and untreated 5-8F cells, and thirty-three non-redundant proteins were identified. Among them, 15 proteins including nm23-H1 were up-regulated, and 18 proteins were down-regulated after treated with 5-aza-2-dC. Encoding genes of 15 up-regulated proteins may be methylation silenced genes in 5-8F cells; (2) Western blot analysis showed that the expression of nm23-H1 was up-regulated in 5-8F treated with 5-aza-2-dC, which was consistent with the result of proteomic analysis; (3) MSP showed that methylation level of nm23-H1 promoter was decreased, while unmethylation level of that was increased after treated with 5-aza-2-dC, and RT-PCR showed that nm23-H1 mRNA expression was increased obviously after treated with 5-aza-2-dC, which suggested that nm23-H1 is methylation silenced gene in 5-8F.In this study, it is the first time using proteomic analysis to screen for methylation silenced gene in NPC. 15 candidate methylation silenced genes in NPC cells were identified, and our data suggested that nm23-H1 is a methylation silenced gene in NPC cells, which may play a critical role in the pathogenesis of NPC. The results will be helpful for further investigating methylation silenced genes in NPC, and provide a new idea for screening methylation silenced genes involved in the pathogenesis of tumor.