锌指蛋白83(ZNF83)抑制膀胱移行细胞癌增殖迁移的功能和机制研究

The incidence and mortality of bladder cancer ranks the first of the urinary system tumors，transitional cell carcinoma（TCC）accounts for more than 90% of bladder cancer. The applicant selected out a high-frequency mutation ZNF83 in the large sample genomic research of TCC. The gene encodes human zinc finger protein 83. Zinc finger protein is a type of transcription factor, whose abnormal expression plays an important role in malignant tumor proliferation and metastasis. There is no study reported about the functions and mechanisms of ZNF83 till now. Our preliminary experiments data shows that the mutation of ZNF83 was associated with the poor prognosis of TCC patients, ZNF83 had a low expression in TCC cell lines, and the overexpression of ZNF83 inhibited cell proliferation. These data suggests that ZNF83 plays the role of tumor suppressor gene by transcriptional regulation in the proliferation and migration of TCC. This project intends: 1. To further study the association between ZNF83 mutation and the clinical features；2. To study the function of ZNF83 in TCC evolution and progression by the lentivirus transfection, PCRsanger sequencing, cell function experiments in vitro and animal models tumor formation techniques. 3. To study the molecular mechanisms of ZNF83 regulating the downstream gene to suppress TCC proliferation and migration by ChIP-seq and other methods. This study is expected to provide a new target for the diagnosis, treatment and prognosis of bladder cancer.

膀胱癌的发病率和死亡率居我国泌尿系统肿瘤首位，膀胱移行细胞癌（TCC）占膀胱癌90%以上。申请人前期通过TCC大样本基因组测序筛选出一个新的高频突变基因ZNF83，其编码人类锌指蛋白83。锌指蛋白是一类转录因子，其异常表达可促进恶性肿瘤发生发展。目前ZNF83在肿瘤发生发展中的功能和机制尚不明确。本项目预实验显示：ZNF83突变与TCC患者预后较差相关，其在TCC细胞株中低表达，过表达可抑制TCC细胞增殖和迁移。综上推测：ZNF83可通过转录调控下游基因表达，抑制TCC的增殖迁移。本项目拟：1.进一步分析ZNF83突变与TCC临床特征的相关性；2.通过PCRsanger测序、慢病毒转染、细胞功能实验、动物活体成瘤等技术，研究ZNF83在TCC发生发展中的功能；3.通过ChIP-seq等方法探索ZNF83调控下游基因抑制TCC增殖迁移的分子机制。本研究有望为膀胱癌的诊治和预后判断提供新靶标。

目的：移行细胞癌（TCC）是膀胱癌的主要形式。迄今为止，已经获得了关于TCC的遗传基础的大量见解。然而，具有治疗潜力和TCC潜在发病机理的关键驱动子突变基因仍然未知。通过对99个膀胱癌样品进行全基因组测序和外显子组测序，我们确定了一个经常突变的基因ZNF83，其作用尚不清楚。本研究旨在确定ZNF83在TCC发育中的生物学功能和分子机制。.方法：采用全基因组和外显子组序列进行鉴定，采用Sanger序列进行ZNF83突变的验证。在TCC细胞和异种移植模型中研究了致病变体的作用。.结果：在所有病例中，ZNF83在18％的病例中被发现经常发生突变。 E293V在其进化保守域中是突变最多的位点。 ZNF83失活的改变与TCC的高恶性程度和不良预后显着相关。在正常人尿路上皮细胞中使用CRISPR / Cas9系统敲除ZNF83可以显著增加肿瘤的生长，而ZNF83-E293V的引入则发挥了负面作用，降低了TCC中野生型ZNF83的抑癌活性。机制研究表明，功能丧失的ZNF83-E293V突变导致S100A8的转录抑制功能受损和NF-κB信号传导异常，从而导致膀胱癌。.结论：本研究发现ZNF83经常发生突变，并通过调节TCC中的S100A8-NF-κB轴而成为一种新型的肿瘤抑制因子，从而支持ZNF83作为TCC干预的潜在靶点