异构酶Pin1启动子多态性与鼻咽癌的关联分析及其表达调控机制研究

Based on our previous findings that isomerase Pin1 overexpressed in nasopharyngeal carcinoma (NPC) tissues,promoted NPC cells malignant transformation and its promoter polymorphism exhibited a relevance with NPC occurrence risk,this project will focus on studying the relevance between Pin1 promoter polymorphism and NPC occurrence risk,grade malignancy,and identification the transcription factors which regulate the Pin1 promoter. Restriction fragment length polymorphism (RFLP) method will be used for detection the Pin1 two sites（-842 G>C and -667 T>C）single nucleotide polymorphism (SNP)　in 1000 NPC patients and healthy people；The regulated expression of Pin1 itself and the relationship between grade malignancy of NPC and Pin1 expression level will be studied by transfection the plasmids of luciferase reporter system and polymorphism sites of Pin1 promoter; The candidates of transcription factors targeted on Pin1 will be selected by using bioinformatics prediction methods and chromosome co-immunoprecipitation (ChIP). Once this application is approved, we will clearly clarificate the relationship between Pin1 promoter polymorphism and NPC occurrence risk,clinical stages and Pin1 itself expression mechanism. All findings will provide molecular markers for NPC early diagnosis,grade malignancy precaution,prognosis and clues for subsequent research works.

基于本课题组已发现异构酶Pin1在鼻咽癌（NPC）组织中异常高表达、促进NPC细胞恶性转化及小样本分析提示Pin1启动子多态性与NPC发生风险可能相关的基础上,本项目拟采用大样本病例研究Pin1启动子多态性与NPC发生风险以及恶性程度的相关性，并筛选调控Pin1启动子的转录因子。采用限制性片段长度多态性（RFLP）方法检测1000例NPC和健康人Pin1两个SNP位点（-842G>C和-667T>C）基因型及组织检查Pin1表达情况，分析这两位点多态性与发病风险及恶性程度相关性；通过荧光素酶报告基因转染研究位点多态性对Pin1表达变化及对NPC恶性程度的影响；结合生物信息学预测及染色体免疫共沉淀(ChIP)等筛选Pin1结合的转录因子。本课题的完成将明确Pin1启动子多态性与NPC发生风险及临床阶段的关系，阐明Pin1自身表达调控机制，为NPC早期诊断、恶性程度预警提供可靠的分子指标。

基于本课题组已发现异构酶Pin1在鼻咽细胞恶性转化中起作用及Pin1启动子多态性与鼻咽癌发病风险存在相关性的基础上,本课题拟采用大样本病例研究Pin1启动子多态性与鼻咽癌早期发生、进展及预后的相关性,并在鼻咽癌中鉴定出调节Pin1表达的转录因子。采用RFLP方法检测了NPC高发地区的733例鼻咽癌和895例健康人Pin1两个SNP位点（-842G>C和-667T>C）的基因型的荧光素酶报告基因等方法对Pin1多态性在鼻咽癌中如何影响Pin1表达及活性的机制进行研究。我们发现了Pin1启动子（-842G>C和-667T>C）两位点的多态性决定了Pin1启动子活性高低。且最新发表在Sci Rep.上。在该课题资助下还发现Pin1调控ATF1促进鼻咽细胞癌变的规律，尤其是新发现了ATF1的磷酸化位点Thr184也是Pin1的结合位点和维持ATF1稳定功的作用，发表在Cell Death Dis. 2016 Dec 29;7(12):e2571. doi: 10.1038/cddis.2016.349.PMID: 28032861。本课题的完成明确了Pin1多态性与鼻咽癌临床阶段的关系及Pin1在鼻咽细胞癌变中的作用及分子机制，并为鼻咽癌早期发生、进展及预后提供临床辅助诊断的关键性靶分子提供实验依据，作为“一种鼻咽癌发病风险关联的分子检查方法” 的国家发明专利申请已正式受理，目前正在审理中。