MicroRNA-145通过影响RNA甲基化水平促进肝癌细胞抑癌基因表达的作用及其分子机制的研究

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Moreover，it has been reported that miRNA has relationship with HCC. RNA N6-methyladenosine (N6-methyladenosine, m6A) is a modification that is commonly found in higher eukaryotic mRNA. But the role of the modification in cell biological functions is still unclear. YTH domain family 2 (YTHDF2) is a m6A recognition protein that is responsible for identifying the m6A site on mRNA and regulating the stability of mRNA, displaying a vital role in mRNA regulation. Previous study showed that YTHDF2 was closely related to the malignancy of HCC. MiR-145 regulated the expression level of YTHDF2 by targeting the YTHDF2 mRNA 3'UTR in HCC cells to affect the mRNA methylation levels in HCC cells. YTHDF2 is a kind of functional protein which plays important roles in gene regulation. It is predicted that YTHDF2 has many targets including tumor supper genes. Therefore，we deduce that miR-145 could promote tumor suppessor gene expression through down-regulating YTHDF2. In this study, we propose to investigate the critical role of miR-145 on tumor suppessor gene expression through RNA methylation regulation and its molecular mechanism in vitro and in vivo. Our finding provides new insights into the mechanism of RNA methylation in HCC. The new regulation mode of tumor suppessor gene may provide new strategy of HCC clinical therapy .

肝癌是常见的恶性肿瘤，miRNA与肝癌发展有密切的关系。近年来RNA甲基化修饰与癌症发展的关系受到广泛关注。YTHDF2是一种RNA甲基化识别蛋白，在RNA甲基化调控方面发挥重要的作用。我们前期研究发现YTHDF2与肝癌的恶性程度有关，miR-145能够调节YTHDF2影响肝癌细胞中mRNA的甲基化水平。YTHDF2的靶基因包含抑癌基因。因此本项目在前期研究基础上进一步提出科学假设，YTHDF2能够通过调节RNA甲基化水平影响抑癌基因的功能，miR-145能够通过调节YTHDF2促进抑癌基因的表达。本项目将在预实验研究的基础上，拟采用体外细胞和体内动物模型深入进行miR-145通过调节YTHDF2影响RNA甲基化水平促进肝癌细胞抑癌基因表达的研究。本研究将进一步阐明RNA甲基化调控在肝癌中发挥的重要作用，重要抑癌基因的调控方式将对肝癌的治疗提供新思路，具有重要的理论意义和实际应用价值。

RNA甲基化m6A（N6-methyladenosine）修饰大量存在于mRNA中，可以通过调节mRNA稳定性和可变剪接影响mRNA的命运，进而通过调节基因的表达影响肿瘤的发生发展。本研究旨在通过生物信息学挖掘及实验验证探讨m6A调控在肝癌和结直肠癌症发生发展过程中的重要作用。研究发现m6A识别蛋白YTHDF1能够通过相分离现象促进miRNA对抑癌基因的调控作用进而促进肝癌细胞的增殖。YTHDF1能够结合抑癌基因mRNA的m6A位点，进而引发相分离现象，招募沉默复合物RISC帮助miRNA发挥抑制作用，抑制抑癌基因的表达，进而促进肝癌细胞的增殖。该研究揭示YTHDF1在调控肝癌的发生发展中扮演的重要角色，并发现其引发的相分离现象能够参与m6A调控的分子机制，为后续研究m6A与相分离之间的联系在癌症发展中的作用打下良好基础。进一步探究了其它m6A相关蛋白在不同癌症类型中调节的广泛作用，并发现RBM15（m6A甲基转移酶成员）在结直肠癌组织中表达水平升高，RBM15表达量的上调会增加TRIM6的m6A修饰水平，加强m6A识别蛋白IGF2BP3识别TRIM6 mRNA进而增强TRIM6 mRNA的稳定性，导致TRIM6蛋白水平的积累。TRIM6作为促癌蛋白，它的异常升高能够促进结直肠癌细胞的增殖及迁移。以上研究结果揭示了m6A调控相关蛋白在肝癌与结直肠癌的发生发展过程中发挥的重要作用，通过阻断促癌m6A调控相关蛋白发挥功能有望成为治疗癌症的新思路，具有重大的理论研究意义和实际应用价值。