基于转座子诱变模型筛选肾癌发生相关lncRNA及其作用机制研究

The deregulation of several genes, such as VHL, is believed to be responsible for renal cell carcinoma (RCC) tumorigenesis and development. However, knockout of VHL in mice are not predisposed to initiate RCC automatically. Therefore, it is needed to further explore the critical molecular events driving RCC tumorigenesis. In previous study, we successfully established autochthonous mice RCC model through Sleeping Beauty (SB) transposons mutagenesis. Sequencing of transposon insertion sites from mice kidney tumors, followed by functional CRISPR-Cas9 library screen, we identified two driver lncRNAs for RCC tumorigenesis -- lncARC and lncDRC. In this project, we will establish lncARC or lncDRC overexpression/knockout stable cell lines using CRISPR-Cas9 technique, and investigate their roles in the malignant transformation of RCC both in vitro and in vivo. Furthermore, lncARC lsl/lsl and lncDRC fl/fl transgenic mice will be developed to validate their roles in RCC development. Moreover, high-throughput screening such as ChIRP-seq and mass spectrum, as well as classic molecular biology assays will be used to determine the direct target and downstream signaling of lncARC and lncDRC. We will also evaluate the potential of lncARC and lncDRC as potential biomarkers for RCC diagnosis, prognosis and therapy.

VHL基因的失活突变被认为是肾癌发病的主要机制，但VHL敲除的小鼠并不产生肾癌，提示可能存在协同VHL突变促进肾癌发生的关键分子。项目组前期利用睡美人转座小鼠模型成功诱导出小鼠肾癌发生，通过对肾肿瘤中转座插入位点的高通量筛选和基于CRISPR-Cas9文库的功能水平验证，发现了2个介导肾癌发生的关键长链非编码RNA-lncARC和lncDRC。本项目拟进一步利用CRISPR-Cas9技术构建过表达/敲除lncARC和lncDRC的细胞系，研究其对肾癌恶性转化的影响；构建lncARC条件性过表达小鼠和lncDRC条件性敲除小鼠，通过转基因小鼠模型进一步验证其对肾癌发生的调控作用；通过ChIRP-seq、质谱等高通量筛选与经典分子生物学技术，探明lncARC和lncDRC的互作分子及下游信号通路；利用大规模临床肾癌组织和血清样本，评估二者在肾癌预后判定、用药指导和血清学诊断方面的应用价值。

肾癌是泌尿系统最常见的肿瘤之一，近十年来其发病率和死亡率越来越高，成为全球面临的重要健康问题，因此利用睡美人转座小鼠模型对肾癌进展相关的关键基因进行筛选，有助于肾癌发生发展机制的深入了解。我们根据项目计划书要求，分阶段开展了课题研究，并在本项目支持下拓展了研究内容，①完成了lncRNA调控肾癌发生发展的生物学机制研究。②系统分析了肾癌组织中的lncRNA谱，结合睡美人转座系统筛选出的关键lncRNA，建立并验证了能够准确有效评估早期肾癌患者预后的lncRNA模型。③阐述了肾脏肉瘤发生发展的生物学机制。④提出了肾癌索拉非尼耐药的新机制。从肾脏肿瘤发生发展的生物学机制，晚期肾癌耐药机制，肾癌患者预后评估模型三大方面，为肾癌的精准诊疗提供了新的思路和策略。