YTHDF2通过m6A修饰的AMOTL2调控前列腺癌增殖及上皮间充质转化的分子机制研究

N6-methylated-adenylate (m6A) modification is the most common methylation for RNA, which is considered playing important roles in many physiological and pathological processes in tumors. However, its function is still unclear in prostate cancer. YTHDF2 is an important binding protein for m6A modification. In our previous experiments, knock down of YTHDF2 significantly inhibited proliferation and metastasis of prostate cancer cell lines. AMOTL2 was identified as a potential target of YTHDF2 by MeRIP high-throughput sequencing. This project intends to determine the regulatory relationship and specific mechanism of YTHDF2, AMOTL2 and m6A modification levels in prostate cancer by clinical sample analysis, RIP, RNA-pull down and other experiments. The effects of YTHDF2-regulated AMOTL2 on prostate cancer proliferation and metastasis will be revealed at molecular, cellular, tissue levels and animal models. The key signaling pathway for the downstream effects of YTHDF2-AMOTL2 regulation will be explored. This project has important practical significance for further exploring the new mechanism of prostate cancer and finding new targets for cancer prevention.

RNA的N6-甲基化-腺苷酸（m6A）修饰近年来被认为广泛参与肿瘤发生发展的各个阶段，但其在前列腺癌中的具体作用仍不清楚。YTHDF2是m6A修饰重要的结合蛋白。申请人前期实验显示，下调YTHDF2能够显著提升前列腺癌细胞的总m6A修饰水平，抑制前列腺癌细胞增殖及迁移；通过MeRIP-Seq及转录组测序等技术，初步筛选出YTHDF2调控前列腺癌生物学行为的关键靶蛋白AMOTL2。本项目拟通过临床样本分析、RIP、RNA pull-down等方法明确在前列腺癌中YTHDF2、AMOTL2及m6A修饰水平间的调控关系及具体机制；在分子、细胞、组织水平及动物模型中揭示YTHDF2调控的AMOTL2对前列腺癌增殖及转移的影响；并深入探索YTHDF2-AMOTL2调控轴下游影响的关键信号通路。本项目对深入探讨前列腺癌发病新机制，寻找抗癌新靶点具有重要的现实意义。

本项目以表观遗传学修饰与泌尿系统肿瘤上皮-间充质（EMT）转化为核心，开展系列研究。.1）N6甲基腺苷（m6A）是人类mRNA中最丰富的修饰。YTHDF2作为重要的阅读蛋白，通常以m6A依赖的方式介导mRNA的降解。但m6A相关基因尤其是YTHDF2在前列腺癌中的作用和机制仍不清楚。我们发现前列腺癌中的高表达YTHDF2和m6A核心基因METTL3的患者总体生存率更差。抑制YTHDF2或METTL3可显著抑制前列腺癌体内外的增殖和迁移。通过RNA-seq、MeRIP-seq和RIP-seq高通量测序结合生物信息学技术，LHPP和NKX3-1被鉴定为YTHDF2和METTL3的直接靶点。YTHDF2直接与LHPP和NKX3-1 mRNA 的m6A修饰位点结合，介导mRNA降解。沉默YTHDF2或METTL3能够显著提升LHPP和NKX3-1 mRNA和蛋白水平，并抑制磷酸化AKT。该部分研究阐明，METTL3/YTHDF2/LHPP/NKX3-1轴通过AKT磷酸化调控前列腺癌增殖及EMT过程。该部分研究为前列腺癌潜在诊断生物标志物或治疗靶点的开发提供了理论基础。.2）miRNAs能够调控泌尿系肿瘤的生物学行为。在本项目延伸研究中我们发现，miR-665作为DLK1-DIO3 miRNA簇的一部分，膀胱癌中表达下调。过表达miR-665能够显著下调SMAD3、磷酸化-SMAD3和SNAIL的表达水平，从而逆转EMT，抑制膀胱癌症细胞的迁移。通过数据库初筛及双荧光素报告基因实验的验证，本课题组发现miR-665与SMAD3的3’-非翻译区直接结合。进一步地，过表达SMAD3可以逆转miR-665对膀胱癌迁移及EMT过程的抑制。这项研究揭示了miR-665/SMAD3/SNAIL轴在膀胱癌进展过程中的重要作用，为临床治疗提供新思路。