METTL3通过介导pri-miR-145的m6A甲基化抑制肾癌恶性进展的机制研究

Epigenetic modification of RNA is an emerging hotspot of cancer study in recent years. METTL3 (M3) was verificated and screened from RNA methyltransferase family by our project team, and acts as a potential tumor suppressor gene with significantly lower expression in renal cell carcinoma (RCC) tissue. Our previous study showed that: ①M3 has a significantly negative correlation with tumor size and clinical stage, and its higher expression indicates better prognosis. ② In vivo and in vitro experiments show that M3 could inhibite the malignant phenotype of invasion, migration and proliferation in RCC. ③The literature indicated that M3 functions through epigenetic regulation of m6A methylation in precursor of target RNA. Bioinformatics analysis and preliminary experiment showed pri-miR-145 acted as potential regulatory target of M3. Based on the above, we hypothesize that M3 could promote the maturation of tumor suppressor miR-145 through mediating m6A methylation of pri-miR-145, and then suppress the malignant progression of RCC. We plan to confim that M3 could inhibite malignant progression of RCC with studies in cells, animals and clinical samples. The rescue experiment is designed using silence and over expression strategy to clarify the molecular mechanism of the hypothesis that M3 could promote the maturation of miR-145 through mediating m6A methylation of pri-miR-145. There is no report about the function and mechanism of M3 regulating malignant progression of RCC so far. Our study has source innovation, which could provide new ideas for the diagnosis and treatment of RCC.

RNA表观遗传修饰是近几年肿瘤领域新兴热点。METTL3（简称M3）是项目组从RNA甲基转移酶家族中验证筛选获得、显著低表达于肾癌组织的潜在“抑癌”基因。前期发现：①M3与肿瘤大小和分期显著负相关，表达越高预后越好；②体内外实验表明M3可抑制肾癌侵袭迁移和增殖等恶性表型；③文献表明M3主要通过表观调控靶基因RNA前体m6A甲基化实现功能，生物信息学分析及预实验显示pri-miR-145是M3潜在调控靶点。综上提出假说：M3通过介导pri-miR-145 的m6A甲基化促进抑癌miR-145成熟，从而抑制肾癌恶性进展。本研究拟从细胞、动物、临床样本三个层面证实M3可抑制肾癌恶性进展；结合沉默/过表达策略，设计拯救实验，阐明M3介导pri-miR-145的m6A甲基化促进miR-145成熟的分子机制。有关M3调控肾癌恶性进展的功能及机制未见报道，本研究具有源头创新性，可为肾癌诊疗提供新思路。

肾癌是泌尿系统常见恶性肿瘤，手术是主要治疗方式，然而局限性肾癌患者术后仍有20-40%复发率。此外，确诊时较多患者已发生远处转移，肾癌对放化疗不敏感，晚期肾癌患者分子靶向治疗后易耐药。因此，肾癌发生发展的分子机制及潜在治疗靶点有待进一步探索。申请人长期致力于泌尿系肿瘤尤其肾癌研究，RNA甲基化修饰是生物学领域新兴热点，团队早期关注并开展甲基转移酶复合物在肿瘤中研究。本研究中，采用qRT-PCR检测25对肾癌及对应肾正常组织mRNA中METTL3（简称M3）表达，发现肾癌中M3表达明显降低；采用Western Blot检测7对肾癌及肾正常组织M3蛋白表达，发现M3在肾癌中明显低表达。并发现M3与肿瘤大小和分期显著负相关，表达越高预后越好。单变量分析显示，肾癌患者预后时间明显与年龄、肿瘤大小、病理分期、肿瘤分级和M3表达有关；多变量分析显示，年龄、病理分期、肿瘤分级和M3表达是肾癌患者预后的独立预测因素。检测肾癌细胞系（CAKI-1，CAKI-2，ACHN）及对照肾小管上皮细胞系（HK-2）中M3表达情况，发现mRNA及蛋白水平M3均明显低表达。采用慢病毒稳转CAKI-1和CAKI-2细胞系，嘌呤霉素筛选，直至培养出稳转M3敲低和过表达细胞系，并通过RT-PCR 及Western Blot验证。CCK-8、单克隆细胞增殖、细胞迁移/侵袭、细胞划痕及细胞周期实验证实M3可抑制肾癌侵袭、迁移和增殖等恶性表型。并检测mTOR及EMT通路中蛋白，验证相关通路蛋白变化。挑选CAKI-1细胞行裸鼠皮下成瘤实验，结果提示过表达M3后能抑制瘤体生长。通过生信分析，筛选出肾癌中4个差异表达的M3潜在调控的miRNAs。进一步敲低和过表达M3后，筛选出pri-miR-145和miR-145为靶向基因。由于miR-145在多种肿瘤中抑癌作用已得到证实，未行后续验证。该研究为评估肾癌患者预后及指导个体化治疗提供理论基础。因为新冠疫情及实验室因素，后续研究有一定调整。团队同时探索肾癌发生发展危险因素，免疫微环境变化，治疗方式选择及预后评估等。课题期间，共发表SCI论文7篇，授权专利8件，软件著作权1件，并主编英文书籍2本，副主编英文书籍1本。