CDCP1 m6A RNA甲基化调控在膀胱癌发生发展中的作用及其机制

N6-methyladenosine (m6A) regulation is an important content of RNA epigenetics. It becomes new hotspot in life science. m6A, as a novel regulator for RNA metablism, is invovled in cancer development. However, the relationship betwent m6A and bladder cancer is unclear. In our previous studies, we first found CUB domain-containing protein1 (CDCP1), as a key molecule for cell signaling pathway in cancer, was regulated by m6A modifications, and high expression of CDCP1 in bladder cancer cells enhanced cells migration. However, biological function and mechanism of CDCP1 and its mRNA m6A modification in bladder cancer development have remained obscure. In this project, we will further identify relationship among enzymes for m6A modification, CDCP1 expression and CDCP1 m6A in bladder cancer. Moreover, we will investigate function and molecular mechanism of m6A modification enzymes and CDCP1 m6A in the occurrence and development of bladder cancer by gain-of-function,loss-of-function and rescure strategy. Furthermore, we will explore effects of targeting m6A modification of CDCP1 on bladder cancer development by RCas9 technology. This project will not only lay an important theoretical foundation for revealing molecular mechanism of bladder cancer RNA epigenetics, but provide a novel strategy for targeted intervention of bladder cancer.

RNA腺嘌呤甲基化(m6A)调控是RNA表观遗传学重要研究内容，亦是生命科学新的研究热点。m6A作为一种新的RNA代谢调控因子参与肿瘤发生发展过程，但其与膀胱癌的相关性尚未见报道。我们前期研究首次发现人膀胱癌细胞中肿瘤关键信号通路分子--CUB结构域跨膜糖蛋白1（CDCP1）存在m6A修饰调控，且蛋白表达升高，并促进膀胱癌细胞的迁移。但CDCP1在膀胱癌中作用及其m6A调控机制尚未明确。本项目拟进一步阐明膀胱癌中m6A修饰酶、CDCP1表达及其m6A修饰之间的相关性；通过基因功能获得、缺失及拯救实验策略，揭示m6A RNA甲基化修饰酶及其介导CDCP1 m6A修饰在膀胱癌发生发展中的作用和调控机制；进而利用新型的RCas9技术探索靶向CDCP1 m6A修饰对膀胱癌发生发展的影响。本项目不仅为揭示膀胱癌RNA表观遗传学机制提供重要的理论基础，而且为膀胱癌靶向干预提供新的策略。

RNA腺嘌呤甲基化(m6A)调控是RNA表观遗传学重要研究内容，亦是生命科学新的研究热点。m6A作为一种新的RNA代谢调控因子参与肿瘤发生发展过程，但其与膀胱癌的相关性尚未见报道。我们前期研究首次发现人膀胱癌细胞中肿瘤关键信号通路分子--CUB结构域跨膜糖蛋白1（CDCP1）存在m6A修饰调控，且蛋白表达升高，并促进膀胱癌细胞的迁移。本项目首先探讨了METTL3-m6A-CDCP1信号轴在膀胱癌发生发展中作用。研究发现METTL3和CDCP1在膀胱癌组织中表达升高，并与膀胱癌的进展相关；体内体外功能实验证实METTL3-m6A-CDCP1信号轴促进膀胱癌细胞增殖、侵袭迁移；重要的是，我们创新性构建Rcas9-m6A靶向修饰系统这一新技术，并通过METTL3CD-RCas9系统靶向修饰CDCP1 RNA m6A，增强其翻译从而促进膀胱癌进展。同时，我们研究发现整合素α6（ITGA6）是METTL3或ALKBH5调控的下游分子之一，膀胱癌细胞中ITGA6 mRNA 3′UTR上m6A位点受到METTL3或ALKBH5的调控，YTHDF1/YTHDF3结合ITGA6 mRNA 3′UTR上m6A促进其mRNA的翻译，使ITGA6蛋白水平升高。敲除ITGA6抑制细胞黏附、增殖、迁移、侵袭能力、肿瘤球的形成能力以及体内肿瘤生长及转移能力。过表达ITGA6可以回复METTL3敲除引起的细胞黏附、侵袭迁移表型。上述结果表明CDCP1和ITGA6 mRNA的m6A修饰在膀胱癌的发生发展过程中起重要的促进作用；Rcas9技术可靶向调控m6A修饰。本研究不仅为揭示膀胱癌RNA表观遗传学机制提供重要的理论基础，而且为膀胱癌靶向干预提供新的策略。