mRNA m6A 修饰调控肝癌上皮间质转化发生的表观机制

Epigenetic regulation during carcinogenesis remains focus of cancer research. Hepatocellular carcinoma (HCC) morbidity is very high in China, which severely threaten people’s life. As an important contributor to cancer metastasis, EMT and the underlying mechanisms deserve much attention, which may provide new ideas for cancer diagnosis and treatment. m6A( N6-methyladenosine) is ubiquitous in mRNA of eukaryon and involves in pathogenesis of diseases such as development defects, neuron disorders, immunodeficiency and cancers by regulating mRNA splicing, stability or translation efficiency. In our previous study, we conducted meRIP-Seq of HCC tissues occurring EMT and the para-carcinoma tissues. The results showed that the m6A modification level was much higher in EMT HCC and transcripts uniquely m6A-modified in EMT HCC were clustered in cell adhesion. Whether m6A exerts its impact on stability or translation efficiency of target mRNA will be detected in an in vitro model mimic EMT, which is fulfilled by adding TGF-β to HCC cell line to induce EMT. Furthermore, the transcription factor SMAD3 downstream of TGF-β will be tested by PAR-CLIP to confirm its involvement in the selection specificity of m6A target.

上皮间质转化（EMT）作为促进癌症转移的重要因素，对其发生机制的深入探索可为设计新的诊断、治疗方法及用药提供理论基础。N6-甲基腺苷（m6A）修饰在真核生物mRNA中广泛存在，通过调控RNA代谢参与干细胞干性维持及分化、生物钟节律调节、免疫应答及肿瘤发生发展等重要生理病理过程。本项目前期对发生EMT的肝癌及对应癌旁组织进行甲基化RNA免疫沉淀（meRIP）测序，表明发生EMT的肝癌其m6A修饰水平显著高于癌旁，且主要聚类在细胞黏附上。后续拟通过TGF-β刺激肝癌细胞系模拟EMT发生过程，一方面通过降解率测定及核糖体谱分析明确m6A修饰靶分子稳定性及翻译效率的变化规律及其参与EMT发生的潜在机制，另一方面通过免疫双荧光联合PAR-CLIP技术明确TGF-β下游重要转录因子SMAD3是否可招募并结合m6A甲基转移酶复合体，参与肝癌EMT过程m6A靶分子的特异性选则。

肝癌（Hepatocellular cancer）转移是晚期HCC病人总体预后差，5年生存率低于10%的主要原因。从传统的化学疗法、放射疗法到近现代的靶向疗法、免疫疗法，对于肝癌发生机制、转移机制、肿瘤微环境认识的加深正在逐渐加深我们对于肿瘤的认识。然而，治愈肿瘤包括肝癌的任务依然艰巨，新的靶向分子、药物、治疗手段仍需要不断开发。本研究中，PCDHGA9被定性为肿瘤转移抑制基因。体外PCDHGA9稳定敲低肝癌细胞系shPCDHGA9-SK-HEP-1和shPCDHGA9-HepG2具有非常显著地促进肿瘤细胞迁移的作用；体内腹腔和经脾接种shPCDHGA9-SK-HEP-1细胞的荷瘤小鼠进一步证明此结论，其肝脏被大体积肿瘤占据，腹腔膜、肾脏均有累及，而对照小鼠肝脏呈现散发、多位点、小体积肿瘤。机制上，蛋白组测序及TGF-β诱导细胞EMT实验表明shPCDHGA9-SK-HEP-1具有促进肝癌EMT的作用，并发现TGF-β不影响PCDHGA9转录，但可下调PCDHGA9 mRNA及蛋白水平。MeRIP-qPCR表明TGF-β刺激上调潜在由SMAD3结合METTL3复合体介导的PCDHGA9 m6A修饰并促进其mRNA降解。总结起来，项目提出了通过抑制EMT而抑制肝癌转移的基因PCHGA9，并阐明TGF-β通过上调PCDHGA9 m6A修饰促进其降解从而促进肝癌转移。后续可通过大数量肝癌组织样品进一步验证PCDHGA9的作用，从而判定其是否具有临床转化价值。