METTL14通过m6A甲基化修饰lncRNA-XIST抑制结直肠癌增殖及转移的机制研究

N6-methyladenosine (m6A) is the most predominant modification of messenger RNA in eukaryotic cells and plays a vital role in RNA translation, splicing and stability. Recent studies demonstrated that m6A methylation can also modulate long non-coding RNAs. Nevertheless, the effects of m6A modification on lncRNA function and the underlying mechanisms still remain to be explored. In the present study, we discovered that methyltransferase-like 14 (METTL14), a key m6A methyltransferase, acted as a tumor suppressor by inhibiting proliferation and migration of colorectal cancer (CRC) in vitro and in vivo. The molecular events behind this effect of METTL14 was the m6A methylation and subsequent degradation it mediated on lncRNA-XIST, a potent tumor driving factor of CRC. Moreover, the expression of METTL14 in colorectal cancer was significantly down-regulated and was associated with worse recurrence free survival (RFS) of patients, which suggested METTL14 could be a potential marker of long term survival of CRC. Collectively, we investigated the function, mechanism and potential clinical significance of METTL14 in CRC for the first time, implying the importance of m6A modification in the initiation and progression of colorectal cancer. In addition, we plan to analyze the alteration of downstream signaling resulted from m6A methylation on XIST with mass spectrum, laying foundation to further research.

腺苷酸m6A甲基化是信使RNA（mRNA）最常见的修饰方式，在调控mRNA的翻译、稳定性及剪接方式中发挥重要作用。近期研究表明，长链非编码RNA（lncRNA）也可以发生m6A甲基化修饰，但m6A修饰对lncRNA生物学作用的影响及机制未明。本研究前期工作发现，m6A甲基转移酶METTL14能够在体外及体内抑制结直肠癌细胞增殖和转移，发挥抑癌基因作用。其机制可能是介导肿瘤驱动因子lncRNA-XIST的m6A甲基化修饰及降解。此外，METTL14在结直肠癌中表达水平的降低预示着患者较差的无病生存期，提示METTL14是有潜力的结直肠癌预后标记物。综上，本课题组对METTL14及其介导的m6A甲基化在结直肠癌中生物学作用、分子机制及潜在的临床应用价值进行了首次探索，并计划分析lncRNA-XIST发生m6A甲基化后下游分子及信号通路的改变，为进一步研究指明方向。

近年来RNA的表观遗传修饰引起了研究人员的广泛关注，其中腺苷酸甲基化（m6A）是最常见的RNA修饰方式，两种甲基转移酶METTL3和METTL14能够形成二聚体复合物，在辅助蛋白WTAP的帮助下，完成对信使RNA（mRNA）的腺苷酸甲基化修饰，从而影响mRNA的稳定、翻译、剪接等生物学过程。进而在胚胎发育、干细胞特性及肿瘤发生发展的调控中发挥巨大作用。既往研究表明，METTL3及METTL14主要是对mRNA进行甲基化修饰，并调控下游分子通路。最近有报道称，m6A介导的甲基化也发生于非编码RNA，如microRNA，lncRNA，但机制未明。本项目首先探索了甲基转移酶家族在结直肠癌中的表达水平与临床意义，结果显示METTL14在肿瘤组织中表达降低提示较差的无复发生存期。此外，我们通过功能获得/丧失实验及裸鼠皮下成瘤实验，阐明了METTL14抑制癌细胞增殖、转移的作用，此外，通过高通量测序和RNA甲基化沉淀实验（MeRIP），我们发现METTL14直接结合lncRNA-XIST，促进其降解，进而抑制结直肠癌的增殖和转移，初步阐明了METTL14的作用机制，此外，m6A甲基化是一个动态、可逆的过程，本课题组还初步探索了在此生物学过程中发挥作用的去甲基化酶，预实验结果表明去甲基化酶FTO在结直肠癌中表达升高，可逆转METTL14介导的XIST甲基化，促进结直肠癌转移。综上，本项目探索了RNAm6A甲基化在结直肠癌中的生物学作用及分子机制，初步构建了m6A调控网络，为结直肠癌的治疗提供了新的预测标记物和治疗靶点。