基于定量蛋白质组学技术的铬致细胞恶化转化中SET的潜在作用分析

Analysis of the potential role of SET in chromium-induced malignant transformation cells based on quantitative proteomics

目的分析六价铬[Cr(Ⅵ)]诱导人支气管上皮样细胞(16HBE细胞)恶性转化中蛋白表达谱改变及差异蛋白SET的表达水平,为研究Cr(Ⅵ)致癌的分子机制提供新的线索.方法以Cr(Ⅵ)诱导恶性转化的16HBE细胞为研究对象,提取总蛋白并经烷基化、脱盐,最后酶解为肽段,用Tandem Mass Tag(TMT)进行标记,利用液相色谱-电喷雾质谱(LC-ESI-MS/MS)对标记肽段进行鉴定及相对定量;通过Western blot验证差异表达蛋白SET,并检测其下游分子的表达水平.利用在线分析网站Https://david.ncifcrf.gov对差异蛋白进行基因富集分析.结果 LC-ESI-MS/MS共鉴定3 517个蛋白,上调蛋白185个、下调蛋白201个;基因富集分析发现,差异蛋白主要参与细胞自噬、DNA损伤修复、RNA加工等多个生物学过程;Western blot结果显示,与对照组比较,SET蛋白表达水平上调(P<0.05),SET下游分子H3K18ac、H3K 27ac和p53的表达水平均呈下降趋势,差异均有统计学意义(P<0.05).结论 Cr(Ⅵ)诱导16HBE细胞恶性转化过程中涉及多个生物学过程的蛋白发生改变,SET可能通过抑制H3K18ac、H3K 27ac水平介导p53转录活性的调控模式在Cr(Ⅵ)致癌中发挥重要作用.

Objective To investigate alteration of proteins profile in malignant transformation bronchial epithelial cells(16HBE-T) induced by hexavalent chromium[(Cr(Ⅵ))] and analyze the expression level of SET protein,then to provide some new insights for the carcinogenesis mechanism of Cr(Ⅵ).Methods Total protein was extracted from 16HBE cells and was alkylated and desalinated before digested into peptides.The products were labeled with Tandem Mass Tag (TMT) and identified using LC-ESI-MS/MS.Results A total of 3 517 proteins were found,expression differences greater than 1.5 or less 0.67 times were to found have 185 and 201 proteins,respectively.Gene enrichment analysis revealed that differential proteins were mainly involved in autophagy,DNA damage repair,RNA processing and other biological processes.Western blot results showed the expression level of SET was significantly increased while downregulated in histone H3K18/27 acetylation and p53 protein.Conclusion Proteins involved in multiple biological processes altered in 16HBE-T cells and regulation mode of SET inhibiting histone H3K18/27 acetylation regulating transcriptional activity of p53 may paly an important role in Cr(Ⅵ)-association carcinogenesis.