Quantitative Proteomics Screening Of Nasopharyngeal Carcinoma Metastasis-related Methylation Inactivation Genes

Objective:Quantitative proteomics screening metastasis-related methylation inactivation genes of NPC (nasopharyngeal carcinoma) in panoramic view of the angle, which provide us an important theoretical and scientific basis to reveal the mechanisms of invasion and metastasis of NPC, prevent and control the development of NPC, as well as to improve treatment effects and survival rates of patients with NPC.Methods:MTT assay and flow cytometry were used to determine the optimum concentration of demethylating agent5-aza-2’-dC, which was used to treat5-8F cells with high metastatic potenial. Isobaric tags for relative and absolute quantification (iTRAQ) was performed to separate three kinds of protein samples(treated5-8F cells, untreated5-8F cells and6-10B cells).2DLC-MS/MS was used to identify the differentially expressed proteins. SPSS13.0statistical software was used for the data analysis.Results:The result of MTT assay and flow cytomtry show that the concentration and the treated time of5-aza-2’-dC is5μmol/L and four days respectively, which have a most obvious effect on cell proliferation, apoptosis and cycle arrest. We received a total of49876mass spectro-metry peptide fragments and40496proteins were searched by proteinpilot software, we currently found7kinds of differential proteins. By the MS analysis of relative protein expression of untreated5-8F cell and6-10B cell, we found7kinds of differentially expressed proteins, which were significantly decreased untreated5-8F cell line. By the MS analysis of relative protein expression of untreated5-8F cell and treated5-8F cell, we found that two of seven kinds of down-regulated proteins which was originally decreased in untreated5-8F cell line, Serum albumin and Nuclear pore complex protein Nup85, obtained recovery of expression of protein in treated5-8F cell line.Conclusions:1:5-aza-2’-dC can inhibit the proliferation of nasophar-yngeal carcinoma5-8F cell line, and induce G2/M phase and S phase cycle arrest and cell apoptosis. And this phenomenon has a certain dose-dependent feature. Treats5-8F cell with5μmol/L5-aza-2’-dC for four days will obtain a optimum effect in affecting cell proliferation, cell cycle arrest and apoptosis.2:We finally found7kinds of differential expression protein compare untreated5-8F with untreated6-10B by iTRAQ technology. These proteins were significantly decreased in untreated5-8F cell line. The result indicate that changes of multiple protein expression involved in NPC invasion and metastasis mechanism.3:iTRAQ technology was used to identify the expression of differential proteins of5-aza-2’-dC treated5-8F cell and untreated5-8F cell. We found nuclear pore complex protein Nup85was remarkable expression increased in treated5-8F cell and this protein belong to the one of the original seven down-regulated proteins. Our findings show that down-regulated which mainly caused by gene methylation play a important role in the mechanism of NPC metastasis. However, we need a further study to elaborate specific mechanism.