肺癌酪氨酸硝基化蛋白质靶的系统研究

Lung cancer remains the number-one cancer incidence and mortality. The ability to make an early diagnosis would be of high value to the patient because it would allow for early therapeutic intervention. Oxidative stress is extensively associated with lung cancer. The chemical stable tyrosine nitration is the marker of oxidative injuries and can alter protein’s functions. Evidence demonstrates that the up-stream molecules that elicit protein tyrosine nitration are extensively associated with the etiology of lung cancer including tumor immunity, angiogenesis, and metastasis; that the specific tumor hypoxic microenvironments also contribute to protein tyrosine nitration; and that nitrotyrosine immunoreactivity is significantly increased in tumor-bearing regions relative to non-tumor regions, and in sera of lung-cancer patients relative to controls. However, virtually nothing is known about the protein targets of tyrosine nitration in lung cancer. The overall objective of this program is to identify those proteins that are the targets of tyrosine nitration in human lung cancers, to identify the lung cancer-related nitroproteins, and to discover reliable biomarkers, with proteomics.. The total proteins from tissue will be extracted (cancer; controls). Two-dimensional difference in-gel electrophoresis (2D-DIGE)-based comparative proteomics will be used to determine differential nitrotyrosine proteins (DNTPs); or isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics will be used to determine DNTPs. Tandem mass spectrometry (MS/MS) will determine the nitrotyrosine sites. Pathway and Protein Domain analyses will evaluate protein functions and domains. The identified DNTPs will be verified with immunoprecipitation, Western blot, and MS/MS. . Those DNTPs could be developed into tissue biomarkers for molecular classification of lung cancer and discovery of therapeutic targets. These findings will improve understanding of lung cancer pathology and develop new reliable molecular biomarkers.

肺癌的发病率和死亡率居人类癌症首位，常因起病隐匿、早期症状不明显而错失早期治疗的良机。筛选可靠的早期肺癌诊断标志物，对肺癌的早期诊断、早期治疗有极其重要的临床价值。酪氨酸硝基化是氧化应激的稳定标志物，它与肿瘤免疫、血管新生和癌转移有着广泛联系；此外，肿瘤低氧微环境促进酪氨酸硝基化。在肺癌组织和血清中硝基化酪氨酸的免疫活性水平显著高于对照组，然而与肺癌相关的酪氨酸硝基化的蛋白质靶标却不清楚。本项目采用定量硝基化蛋白质组学来鉴定非小细胞肺癌组织差异酪氨酸硝基化蛋白质及其分子网络的变异，以期发现用于肺癌的分子分类和可作为治疗靶标的肺癌组织特异的酪氨酸硝基化蛋白质，或得到用于肺癌的早期诊断、分级和进展监测的肺癌特异酪氨酸硝基化蛋白质。这将提高人们对肺癌病理的认识，并为肺癌发展新的可靠生物标志物提供依据。

蛋白质酪氨酸硝基化是氧化应激的稳定标志物，它与肿瘤免疫、血管新生和癌转移有着广泛联系；并且肿瘤低氧微环境也促进酪氨酸硝基化。蛋白质硝基化是肺癌病理生理过程的重要分子事件，然而其硝基化的蛋白质靶标却不清楚。本项目采用定量硝基化蛋白质组学鉴定了肺鳞癌组织和癌旁间的酪氨酸硝基化蛋白质、硝基化位点及其分子网络的变异。结果：(1)双向电泳联合抗硝基化酪氨酸抗体的Western blot实验在肺癌和癌旁间鉴定了110个免疫阳性的酪氨硝基化蛋白质点，并在癌和对照见对免疫阳性点进行了配对比较，质谱鉴定了9个硝基化蛋白质和9个硝基化位点。(2)用非标记的Label free定量蛋白质组学方法，在肺鳞癌和癌旁组织间鉴定了15硝基化蛋白质和硝基化位点。这些鉴定的硝基化蛋白质参与多种功能，如参与细胞周期调节、ATP结合、信号传导、细胞链接、细胞凋亡等多种生物学功能。(3)用Biotin衍生法，即先对胰酶消化的肽段的-NH2基团进行乙酰化封闭，然后将肽段中的-NO2基团还原为-NH2基团并进一步将-NH2基团进行Biotin衍生，最后将含有Biotin的肽段分离出来，进行MS/MS鉴定。已在肺癌组织中鉴定了30个硝基化蛋白质和35个硝基化位点。这些数据构成了目前最大的肺癌硝基化蛋白质组数据库，为深入研究肺癌的分子机制、鉴定有效生物标志物打下了基础，为寻找有效治疗靶标以预防和治疗肺癌提供了科学依据。