应用蛋白质芯片检测复发鼻咽癌蛋白磷酸化表达

Protein microarray analysis on changes in protein phosphorylation in recurrent nasopharyngeal carcinoma

目的 比较复发鼻咽癌（rNPC）和初发鼻咽癌（pNPC）相关蛋白质磷酸化的差异.方法 提取在温州医学院附属第一医院2003年1月至2005年9月行放疗的4例复发鼻咽癌和4例初发鼻咽癌的总蛋白,应用磷酸化抗体蛋白质芯片与提取蛋白进行杂交,检测656种蛋白质磷酸化表达水平,筛选差异磷酸化蛋白并进行聚类.PhosphoSite plus在线磷酸化蛋白数据库分析磷酸化位点及其在信号通路中的作用.免疫组织化学验证差异磷酸化蛋白.结果 复发组鼻咽癌和初发组的蛋白质磷酸化表达不同,筛选出6个差异蛋白.KIT、ATP1A1、Synapsin、SEK1及Histone H2AX磷酸化水平在复发组上调（P=0.007 ～0.048）,而c-Jun下调（P =0.030）.复发组H2AX磷酸化表达0.390（0.175）高于初发组0.290（0.155）,复发组c-Jun磷酸化表达0.625（0.145）低于初发组0.725（0.178）,（均P＜0.05）.其中c-Jun、H2AX、SEK1及KIT蛋白质磷酸化涉及DNA损伤修复能力增强、抑制凋亡及致瘤能力增强,与肿瘤复发有关.结论 蛋白质磷酸化水平的改变可能与鼻咽癌复发机制有关,为临床上放射治疗失败提供全新的策略.

Objective To compare the differential phosphorylation level of proteins between relapsed nasopharyngeal carcinoma（rNPC）and primary nasopharyngeal carcinoma（pNPC）.Methods Total protein was extracted from 4 pNPC tissue and 4 rNPC tissue samples from January 2003 to September 2005.Then it was analyzed by antibody microarray with 656 antibodies.The differential phosphorylation level of proteins was screened and clustering analysis conducted.The phosphorylation status of the protein sites and its functional pathways were analyzed via an online database of PhosphoSite Plus.The protein expressions were detected by immunohistochemistry.Results Relapsed and primary nasopharyngeal carcinomas had differential phosphorylation level of proteins.And 6 differentially expressed proteins were identified.The phosphorylation levels of KIT,ATP1A1,Synapsin,SEK1 and histone H2AX were up-regulated in rNPC （P =0.007-0.048）while c-Jun was down-regulated（P =0.030）.The expression of P-H2AX in rNPC was significantly higher than that in pNPC[0.390（0.175）vs 0.290（0.155）],but p-c-Jun was significantly lower in rNPC than that in pNPC[0.625（0.145）vs 0.725（0.178）]（both P〈0.05）.Among them,the changes in the phosphorylation levels of c-Jun,histone H2AX,SEK1 and KIT might play important roles in the relapse of NPC through improving DNA damage repair ability,inhibiting apoptosis and promoting tumorigenesis.Conclusion The changes of protein phosphorylation may help to explain the recurrent mechanisms of NPC and provide new therapeutic anti-recurrence targets.