METTL3低表达调控m6A修饰对胃癌恶性表型的影响

METTL3 is a methyl-transferase of N6-methyladenine (m6A), which can effect the expressions of genes by catalyzing them to get m6A and is tightly associated with the oncogenesis, progression and metastasis of cancer. In previous study by TCGA database, tissue array and cell lines, the expression level of METTL3 obviously decreased in gastric cancer. It implied that METTL3 might play a role in the malignant phenotype of gastric cancer. Thus, this study will focus on determining the role of m6A regulation by METTL3 through the testing of gastric and para-cancer tissue and cell lines, investigating the influence that m6A modification by METTL3 has on the growth, invasion, migration and drug resistance of gastric cancer cells through in vivo and vitro experiments and figuring out the core pathway molecular regulated by METTL3-caused m6A modification through transcript and m6A sequencing. This study is aim to present the concrete effects that down-regulation of METTL3 has made on the malignant phenotype of gastric cancer and the core genes that regulated by METTL3, in order to offer a new epigenetic target for the clinical therapy of gastric cancer.

METTL3是N6-甲基腺嘌呤（m6A）的甲基转移酶，可催化基因的mRNA获得m6A修饰，从而影响基因表达，与肿瘤的发生、进展及转移密切相关。前期TCGA数据库、组织芯片及细胞系验证结果表明，METTL3在胃癌中表达显著降低,提示METTL3极有可能影响胃癌的恶性表型。因此，本项目拟利用胃癌及癌旁组织和细胞系实验，明确胃癌中METTL3对m6A的调节作用，以及METTL3表达与相关临床病理因素及患者生存期的关联。通过体内、体外实验，明确METTL3所引起的m6A修饰变化对胃癌细胞生长、侵袭、迁移、耐药等恶性表型的影响。通过转录组及m6A测序，寻找胃癌中由METTL3进行m6A修饰调控的核心通路分子。本项目旨在揭示METTL3表达降低通过调节m6A修饰对胃癌恶性表型产生的具体影响，同时揭示受METTL3调控的关键基因，试图为临床胃癌治疗提供新的表观遗传学靶点。

胃癌的恶性转移仍是胃癌患者不良预后的最主要原因，关于胃癌恶性转移的机制研究虽已有长足的进展，但参与调控该过程的关键分子仍有待于深入探索。N6-腺苷酸甲基化（m6A）是近年来被深入研究的一种最为常见的RNA修饰，其关键甲基转移酶蛋白3（Methyltransferase like protein 3, METTL3）是典型的癌基因，然而其对胃癌恶性转移的影响及其直接调控的下游关键分子仍有待于进一步的研究。本项目研究发现，与癌旁组织相比，METTL3在胃癌组织中呈现显著高表达，且与胃癌患者的不良预后密切相关。在胃癌细胞中过表达METTL3可促进胃癌细胞的侵袭与迁移能力。反之，敲减METTL3可抑制胃癌细胞的侵袭及迁移能力。在METTL3过表达及对照组细胞和METTL3敲减及对照组细胞的MeRIP测序结果显示，富含亮氨酸重复蛋白8A（leucine rich repeat containing 8 family member A，LRRC8A）的m6A修饰水平受METTL3直接调控，过表达METTL3时，LRRC8A的表达水平相应增高，而敲减METTL3时，LRRC8A的表达水平相应降低。对METTL3过表达及对照组细胞系的YTHDF1 RIP测序结果中同样发现，过表达METTL3组的LRRC8A捕捉量高于对照组，表明YTHDF1是识别LRRC8A的下游Reader蛋白。在对LRRC8A的表达验证中发现，与癌旁组织相比，LRRC8A在胃癌组织中的表达水平显著升高，且导致了患者的不良预后。此外，体内实验表明，应用METTL3抑制剂STM2457或LRRC8A抑制剂DCPIB均可有效抑制小鼠异种移植瘤的增殖。本项目证明了METTL3是胃癌组织中的典型癌基因，可促进胃癌细胞的侵袭与迁移。同时证明了METTL3直接调控的m6A修饰下游关键基因为LRRC8A。LRRC8A也是关键的促进胃癌进展的癌基因。利用小分子抑制剂有效抑制METTL3和LRRC8A的活性是抑制胃癌恶性表型，改善胃癌患者预后的潜力治疗策略。