m6A RNA甲基转移酶METTL3对结直肠癌肿瘤免疫微环境的调控作用和机制研究

大多数结直肠癌患者耐受免疫治疗，因此亟待研究肿瘤内在分子事件异常与肿瘤免疫应答间的关系。m6A（6-甲基腺嘌呤）修饰是最丰富的RNA修饰，我们前期结果表明，敲除m6A甲基转移酶Mettl3可以抑制小鼠的结直肠肿瘤发生，减少髓系免疫抑制细胞MDSC的募集但增多T细胞浸润。敲除METTL3抑制了肠癌细胞CXCL1的表达和分泌，因此肿瘤对MDSC的募集能力减弱；过表达野生型而不是m6A催化活性缺失的METTL3则可以促进CXCL1表达，并诱导MDSC迁移。RNA-seq和m6A-seq分析发现METTL3可能催化多种转录因子m6A修饰进并调控其表达，进而提高细胞因子包括CXCL1的转录。本课题将基于已搭建的Mettl3肠道特异性过表达小鼠和hCD34+人源化小鼠模型，结合m6A-seq、RIP-seq、RNA-seq和Ribo-seq多组学平台，揭示肠癌m6A修饰异常和肿瘤免疫应答间的重要关联。

Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality in China. The development of CRC involves alterations of CRC-intrinsic events and host immune system, as well as their crosstalk. N6-Methyladenosine (m6A) is the most abundant RNA modification in human and a determinant in posttranscriptional regulation of mRNA splicing, translation and degradation. Recent studies from others and us have demonstrated the functional importance of m6A regulators in tumorigenesis. Nevertheless, it is not clear whether and how alterations of tumor cell-intrinsic mRNA m6A modification can dictate the immune landscape of CRC. Our preliminary data showed that knockout of Mettl3, a m6A writer, inhibited colorectal tumorigenesis in ApcMin/+ mice, reduced MDSCs but increased CD4+ and CD8+ T cells frequency, implying that METTL3 promotes an immune-suppressive microenvironment. As tumor-derived cytokines are critical in linkage of tumor and immune function, we audited the expression of cytokines and chemokines and reduced serum CXCL1 was identified after Mettl3 depletion in ApcMin/+ mice. We further demonstrated that METTL3 promoted MDSC accumulation through CXCL1-CXCR2 axis. Silencing of METTL3 in CT26 and MC38 suppressed CXCL1 mRNA and protein expression. Accordingly, blunted MDSC migration was observed with conditioned medium from CRC cells with Mettl3 depletion and this effect was abolished by addition of recombinant CXCL1 protein or CXCR2 inhibitor. Furthermore, wild type but catalytically dead mutant of METTL3 promoted CXCL1 expression and MDSC migration. However, the difference was negligible after CXCL1 depletion, suggesting that METTL3 promotes MDSC migration via CXCL1/CXCR2 axis. To investigate the mechanism by which METTL3 promotes an immune-suppressive environment, we analyzed RNA-seq and m6A-seq data of CRC cells with or without METTL3 silencing. The results demonstrated that METTL3 promoted m6A modification of transcription factors which are possibly responsible for the transcription of cytokines, including CXCL1. In this proposal, based on established syngeneic mice tumor models, humanized mice models and colon-specific Mettl3 knockin mice models, together with m6A-seq, RIP-seq, RNA-seq and Ribo-seq platforms, we aim to investigate the possible mechanism of action by which METTL3-m6A axis in CRC promotes the immune suppression.