m6A甲基转移酶METTL16在结直肠癌增殖转移中的作用及其机制研究

The diagnosis and treatment of advanced colorectal cancer has become an urgent clinical problem since its poor prognosis. RNA m6A modification plays an important role in colorectal cancer (CRC). We found that METTL16, a novel m6A methyltransferase which was significantly highly expressed in CRC, could regulate the level of RNA m6A modification and promote the proliferation and metastasis of CRC cells. The Venn diagram of RNA-seq and m6A-seq analysis showed that RACK1 could be a target gene of METTL16. Further analysis of TCGA and GEO data showed that RACK1 was also highly expressed in CRC and positively correlated with METTL16 expression, which was verified by our pre-experiment. It was also showed that the m6A binding site was located on Intron7 of RACK1. Hence, we propose our hypothesis that METTL16 promote the proliferation and metastasis of CRC by up-regulating RACK1 through m6A pathway. This study intends to explore the function of METTL16 in colorectal cancer and its relationship with prognosis at multiple levels. Furthermore, its mechanism through targeting RACK1 will be also investigated. This study is original and innovative, which will increase the understanding of the pathogenesis of colorectal cancer and provide new ideas for finding prognostic markers and new therapeutic targets for colorectal cancer.

晚期结直肠癌预后差，其诊治已成为临床亟待解决的难题。RNA m6A修饰在结直肠癌的发生发展中起着重要作用，前期发现新型m6A甲基转移酶METTL16在结直肠癌中显著高表达，能够调控RNA m6A水平并促进肠癌细胞的增殖转移等表型。RNA-seq与m6A-seq联合分析示RACK1 mRNA及m6A水平均受METTL16调控，可能为其下游靶基因。进一步分析公共数据发现RACK1在结直肠癌中亦明显高表达，且与METTL16表达呈正相关，并得到验证。测序结果示m6A结合位点位于RACK1 Intron7上。我们提出科学假说：METTL16可能通过m6A途径上调RACK1促进结直肠癌的增殖转移。本研究拟从多层次探索METTL16在结直肠癌中的功能，并以RACK1为切入点探究其作用机制。本研究具有源头创新性，将增加结直肠癌发病机制的了解，为寻找结直肠癌新型预后标志物和治疗靶点提供思路。

晚期结直肠癌预后差，其诊治已成为临床亟待解决的难题。RNA m6A修饰在结直肠癌的发生发展中起着重要作用，TCGA、GEO数据库结直肠癌数据显示，新型m6A甲基转移酶METTL16在结直肠癌较癌旁高表达，能够调控RNA m6A水平并促进结直肠癌细胞的增殖等表型。课题组检测25例结直肠癌及癌旁样本，验证RNA水平METTL16在结直肠癌较癌旁高表达；且TCGA结直肠癌生存数据显示，METTL16高表达与较短OS相关，收集112例结直肠癌患者中，观察到METTL16高表达与较短OS、PFS相关。为深入研究METTL16调控结直肠癌增殖的机制，RNA-seq与m6A-seq联合分析示RACK1 mRNA及m6A水平均受METTL16调控，可能为其下游靶基因。进一步分析公共数据库TCGA、GEO等发现RACK1在结直肠癌中亦明显高表达，且与METTL16表达呈正相关，并在课题组收集的结直肠癌样本中得到验证。体外细胞回复实验验证在结直肠癌中过表达RACK1能够回复敲低METTL16对RACK1表达的影响，且能回复细胞增殖能力及细胞周期进程。RIP实验、放线菌素实验显示METTL16与RACK1 mRNA直接结合并增加其稳定性。MeRIP实验表明METTL16调控RACK1 mRNA的m6A水平，进而证实科学假说：METTL16可通过m6A途径上调RACK1促进结直肠癌的增殖。此外，研究中课题组发现METTL16亦可通过m6A途径负性调控下游EP300的表达进而影响结直肠癌的增殖的生物学行为。本研究具有较高的创新性，增加结直肠癌发病机制的了解，为寻找结直肠癌新型预后标志物和治疗靶点提供了新的思路。