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目的探讨乳腺癌患者血清中蛋白的表达变化,筛选并分析乳腺癌诊断的生物学标志物。方法采用定量蛋白质组学串联质量标签(TMT)标记技术对68例Ⅱ期乳腺癌患者及62例健康女性的血清蛋白进行检测,并进行蛋白质定量分析,筛选乳腺癌患者血清中发生显著变化的差异蛋白。利用Uni Prot数据库和Proteome Discoverer软件及在线工具STRING对差异蛋白进一步行生物信息学分析,应用蛋白质印迹法和q PCR对变化倍数显著的差异蛋白VIME(上调为健康对照女性的3.918倍)和RAF1(下调为健康对照女性的0.251)进行验证。结果共获得67种差异蛋白,其中26种表达上调,41种表达下调。基因本体论(GO)注释分析和功能聚类分析显示上述差异蛋白与肿瘤的血管生成、新陈代谢进程、生物黏附能力等相关。蛋白质印迹法和q PCR验证结果显示RAF1蛋白和mRNA在Ⅱ期乳腺癌患者血清中的表达水平均低于健康对照女性,而VIME蛋白和mRNA的表达均高于健康对照女性,与筛选结果一致。结论定量蛋白质组学TMT法能有效筛选Ⅱ期乳腺癌患者血清中的差异蛋白,其中VIME和RAF1蛋白有望成为乳腺癌淋巴结转移的候选血清标志物。
Objective To investigate the changes of serum protein in patients with breast cancer and to screen and analyze the biomarkers for the diagnosis of breast cancer. Methods The serum protein of 68 patients with stage Ⅱ breast cancer and 62 healthy women were detected by quantitative proteomics tandem mass labeling (TMT), and the serum protein of patients with breast cancer was screened. protein. The bioinformatics analysis of the differential proteins was carried out by using Uni Prot database and Proteome Discoverer software and online tool STRING. Western blotting and q-PCR were used to detect the differential proteins VIME (3.918-fold up-regulated to healthy controls) and RAF1 Down to 0.251 for healthy controls). Results A total of 67 kinds of differential proteins were obtained, of which 26 were up-regulated and 41 were down-regulated. Gene Ontology (GO) annotation analysis and functional clustering analysis showed that the above differential proteins were related to tumor angiogenesis, metabolic process, bioadhesion and so on. Western blotting and q PCR showed that the expression of RAF1 protein and mRNA in serum of patients with stage Ⅱ breast cancer was lower than that of healthy controls, while the expression of VIME protein and mRNA was higher than that of healthy controls, which was consistent with the screening results. Conclusion The quantitative proteomics TMT method can effectively screen the differential proteins in the serum of patients with stage Ⅱ breast cancer. VIME and RAF1 proteins are expected to be candidate serum markers for lymph node metastasis of breast cancer.