肿瘤抗原OVA12调控p53蛋白表达的生物学功能和作用机制

Identifying new tumor antigens and determining their roles and mechanism in tumorigenesis are critical to the development of molecular targeted therapy. Tumor antigen OVA12 was obtained from a human ovarian cancer cDNA library utilizing the approach of SEREX analysis, which is characteristically highly expressed in tumor cells and tissues. We have demonstrated that OVA12 played a notable role in accelerating tumor development both in vitro and in vivo. By using cDNA microarray analysis and protein 2 D gel electrophoresis combined with mass spectrometry, we found that OVA12 could negatively regulated the tumor suppressor gene p53. However, whether OVA12 affects p53 transcriptional activity? Whether OVA12 regulates the expression of mutant p53?What’s the mechanism by which OVA12 negatively regulates p53? Based on these, we will select the p53 wild type and mutant tumor cells, establish the stable over-expressed and knocking-down cancer cell lines and studying the biological function of OVA12 in regulating the p53 expression. Besides, we will explore to elucidate the mechanism by which OVA12 negatively regulates p53 by using Co-immunoprecipitation and protein microarray assay. We believe that the accomplishment of this project is of great theoretical significance and good prospect of application and will provide the research ideas and targets for the development of novel anticancer drug or biologics, which could be a novel breakthrough of tumor immunity field.

寻找新的肿瘤抗原基因和表达蛋白，明确其生物学功能和作用机制是发展肿瘤靶向治疗的关键。肿瘤抗原OVA12是应用重组cDNA表达文库血清学分析技术筛选得到的一个新的蛋白分子，分子量为12KD。申请人在以往研究中证实肿瘤抗原OVA12在肿瘤细胞（组织）中特征性高表达，具有促进肿瘤细胞增殖的重要生物学功能。应用基因芯片等高通量技术寻找OVA12作用靶点，发现OVA12能负向调控抑癌基因p53表达，但OVA12是否会影响p53蛋白转录活性、是否调控突变型p53蛋白表达及调控p53蛋白表达机制等问题尚未解决。因此，本项目选取p53野生型和突变型细胞株，构建OVA12高表达和干扰细胞系，研究OVA12负向调控p53蛋白的生物学功能，并通过免疫共沉淀及蛋白芯片等技术探讨OVA12调控p53蛋白的作用机制。本项目研究具有重要的理论价值和良好的应用前景，能够为研发新的抗肿瘤靶向药物提供研究思路和作用靶点。

发现新的肿瘤抗原基因，结合分子生物学、蛋白组学技术明确其生物学功能，在揭示其作用机制基础上，由基础免疫研究向临床肿瘤诊疗实践不断延伸，不仅具有重要理论价值，而且能为发现新的肿瘤检测标志物、研发新的肿瘤疫苗和肿瘤免疫治疗药物提供新的的理论依据和研究靶点。.近三年来，申请人在国家自然科学青年基金项目的资助下，对肿瘤抗原OVA12促进肿瘤细胞增殖的作用机制展开初步研究。应用Real-time PCR、Western blot、免疫荧光及免疫组化技术进行检测，结果显示肿瘤抗原OVA12能够负向调控p53蛋白的表达。为明确肿瘤抗原OVA12负向调控p53蛋白表达的作用机制，应用免疫共沉淀方法检测OVA12高表达和干扰细胞系在蛋白酶体抑制剂MG132处理后p53蛋白泛素化水平。结果显示，与对照组相比，OVA12表达降低后，p53蛋白泛素化水平明显降低，而在OVA12高表达后，p53蛋白泛素化水平显著提高。应用真核生物蛋白合成抑制剂放线菌酮处理OVA12高表达和干扰细胞系，应用Western blot技术检测p53蛋白在不同时间点的表达水平，结果显示与对照组相比，OVA12干扰后p53半衰期由40min延长至70min左右，而OVA12高表达后p53半衰期由60min减少至30min左右。.为进一步明确肿瘤抗原OVA12调控p53蛋白泛素化降解的详细作用机制，应用Real-time PCR及Western blot技术检测结果表明OVA12不影响MDM2 mRNA及蛋白的表达水平；免疫共沉淀结果显示OVA12与MDM2无相互作用。应用免疫共沉淀技术结合蛋白电泳及质谱技术，结果显示，与对照组相比，发现OVA12高表达组出现四条特异性条带；分别为breakpoint cluster region protein 1, p53, UBE2R2及 myosin light chain3。其中UBE2R2属于泛素－蛋白酶体系统中E2泛素偶联酶家族，为一种重要的泛素偶联酶。因此，根据质谱结果，我们推测肿瘤抗原OVA12可能为一种新的泛素连接酶，与泛素偶联酶UBE2R2相互作用，共同调控p53蛋白在泛素－蛋白酶体的降解过程。