经典PRC1复合体中的五种CBX蛋白在肺腺癌发生发展中的作用及机制研究

Lung adenocarcinoma is one of the most common malignant tumors in human beings, whereas the mechanism involved in its progression needs to be further explored. Epigenetic regulation plays a key role in tumorigenesis. To explore novel epigenetic factors functionally involved in the progression of lung adenocarcinoma, we analyzed 576 RNA-sequencing data (58 pairs of lung adenocarcinoma and normal tissue samples) obtained from TCGA. The analysis indicated that the expression of CBX2, CBX4 and CBX8 are higher in lung adenocarcinoma than normal tissue, and the expression of CBX6 and CBX7 are lower in lung adenocarcinoma. These five CBX proteins are all important components of the canonical PRC1 complex. Furthermore, their expression levels are related to the progression and prognosis of lung adenocarcinoma. Our experimental results also showed that knockdown of CBX2 results in a significant decrease in the growth, proliferation and invasion of lung adenocarcinoma cells, and a decrease in the growth of the implanted tumors in nude mice. We intend to further investigate the role of these five CBX proteins in the growth and metastasis of lung adenocarcinoma and the mechanisms involved, using experimental methods of molecular biology and genetics. Furthermore, ChIP-sequencing will be performed to discover the distribution of different CBX proteins across the genome of lung adenocarcinoma cells, finding the targets of these CBX proteins. Moreover, immunoprecipitation coupled with mass spectrometry will be executed to clarify interacting proteins of different CBXs. Our study will provide new targets for the diagnosis and treatment of clinical lung adenocarcinoma, and add to the understanding of the diverse functions of different canonical PRC1 complexes.

肺腺癌是人类最常见的恶性肿瘤之一，其发生发展调控机制需更深入探究以寻找新的治疗靶标。表观遗传学调控在肿瘤发生发展中发挥着核心作用。通过分析TCGA肺腺癌RNA-seq数据，我们发现经典PRC1复合体组分CBX2、CBX4和CBX8在肺腺癌组织中高表达，而CBX6和CBX7低表达；而且它们表达水平与肺腺癌进程、预后相关。我们前期在细胞和小鼠水平都证实CBX2具有促进肺腺癌生长以及转移的功能。我们拟进一步利用分子生物学和遗传学等实验手段在细胞水平和动物水平探究不同的CBX蛋白在肺腺癌发生发展中的作用及分子机制；同时以肺腺癌为研究模型，利用ChIP-seq和免疫共沉淀-质谱研究不同CBX蛋白在肺腺癌细胞染色质上分布的异同、形成复合体的差异，解析经典PRC1复合体CBX组分的差异导致不同靶向效应的机制。我们的研究将为临床肺腺癌的诊疗提供新的靶标，为更全面了解经典PRC1复合体的功能奠定基础。

肺腺癌是人类最常见的恶性肿瘤之一，其发生发展调控机制需更深入探究以寻找新的治疗靶标。表观遗传学调控在肿瘤发生发展中发挥着核心作用。通过分析TCGA肺腺癌数据，我们发现经典PRC1复合体组分CBX2、CBX4和CBX8在肺腺癌组织中高表达，而CBX6和CBX7低表达。在体内和体外敲低CBX2均能显着抑制肺腺癌细胞生长和转移。CBX2和EZH2的联合高表达，是肺腺癌预后不良的指标。同时敲低CBX2和EZH2对肺腺癌细胞的生长和转移具有协同抑制作用。在机制上，我们发现CBX2和EZH2通过联合或单独与其靶基因启动子区域结合，下调了PPAR信号通路基因和肿瘤抑制基因。此外，敲低CBX2提高了EZH2抑制剂对肺腺癌的治疗效果。另一方面，我们在A549和H1975细胞中过表达或敲低CBX8，通过MTT、EdU-incorporation实验、划痕实验、transwell实验、小鼠荷瘤实验证实CBX8能够在体内外促进肺腺癌细胞增殖和迁移。随后我们通过免疫亲和纯化和质谱分析，鉴定了与CBX8相互作用的蛋白NKRF。之后我们证实敲低NKRF在体内和体外均能显着抑制肺腺癌细胞生长和转移。这一结果提示CBX8可能通过与NKRF相互作用，调节下游靶基因的表达，从而影响肺腺癌细胞的增殖和迁移。我们的研究揭示了CBX2和CBX8促进肺腺癌进展的生物学功能，为肺腺癌的诊疗提供新的靶点。