RNA m6A甲基转移酶METTL14增加非小细胞肺癌放疗敏感性的机制研究

Radiation resistance is a major problem that severely influencing the effect of treatment. Therefore, clarification of the mechanism underlying radiation resistance is clinically important. Recently, the study reported the N6-Methyladenosine (m6A) modification modulates the pri-miRNA/miRNA process in an m6A-dependent manner. Our previous study showed that miR-99a was closely related to the radiation sensitivity, but the mechanism remains to be elucidated. Overexpression of METTL14 is found in radiation-sensitive A549 cells. The expression of METTL14 was positively related with miR-99a. And the m6A modification site was found in pri-miR-99a. The overexpression of METTL14 enhanced the expression of miR-99a and radiation sensitivity in NSCLC cells. Thus, we speculate that METTL14 enhances the radiation sensitivity by modulating the pri-miRNA/miRNA process in a m6A-dependent manner. This research aims to explore the biological functions of METTL14 in radiation resistance by molecular cytology combined with experimental animal models and clinical tissue specimens;exploring the regulatory effects of METTL14 on radiation sensitivity and the expression of pri-miR-99a/miR-99a and their downstream pathways and illustrating the molecular mechanism of METTL14 in radiation resistance. This study will provide a prospective theory and target for the mechanism of radiation resistance in patients with NSCLC, and lay a foundation for future research of transformational medicine.

放疗抵抗严重制约非小细胞肺癌的治疗效果，探究其机制具有重要意义。研究表明，m6A甲基化修饰能调节pri-miRNA/miRNA的成熟过程。我们前期发现miR-99a与非小细胞肺癌放疗敏感性密切相关，但其机制尚待阐明。进一步研究提示：m6A甲基转移酶METTL14在放疗抵抗细胞较敏感细胞中显著低表达，并与miR-99a表达呈正相关，且pri-miR-99a上存在m6A甲基化修饰位点。过表达METTL14上调miR-99a的表达水平并增加放疗敏感性。由此我们推测METTL14通过m6A修饰促进pri-miR-99a/miR-99a成熟，继而增加放疗敏感性。本项目拟从细胞、动物模型并结合临床标本，分析METTL14对pri-miR-99a/miR-99a成熟过程及其下游通路的调节，阐明METTL14调节非小细胞肺癌放疗敏感性的可能机制。本研究将为临床提供前瞻性的增敏靶点并为后续转化医学奠定基础。

放疗抵抗严重影响非小细胞肺癌患者生存，亟需解决。因此，找寻新的有效的放疗增敏靶点和预后标志物是目前非小细胞肺癌治疗领域的科学研究重点。本课题所产生的研究结果均基于对非小细胞肺癌等恶性肿瘤放疗抵抗及预后标志物的探索。其中本课题组重点关注了本课题立项的核心，即m6A调控非小细胞肺癌放疗敏感性的相关研究。我们首先发现了m6A甲基转移酶METTL14在放疗抵抗细胞较敏感细胞中显著低表达，并与miR-99a表达呈正相关，且pri-miR-99a上存在m6A甲基化修饰位点。过表达METTL14上调miR-99a的表达水平并增加放疗敏感性。随后，我们又在m6A调控非编码RNA稳定性上进行了探索。我们发现m6A修饰通过降低RNA降解率，提高RMRP转录本的稳定性；此外，RMRP还能促进细胞的增殖、迁移和侵袭。在机制上，RMRP通过向TGFBR1启动子招募YBX1来促进TGFBR1的转录并继而调控TGFBR1/SMAD2/SMAD3 通路。此外，RMRP促进肿瘤干细胞的特性和上皮间质转化，继而导致NSCLC对放疗的抵抗。这为本课题未来探索m6A与非小细胞放疗抵抗的调控网络提供了宝贵的经验。最后，本课题组发现甲基化基因在肾透明细胞癌患者中高表达，且与患者生存相关，进一步通过体外实验证实了4个基因表达的表达异常可以作为肾透明细胞癌患者的预后标志物。本研究通过上述研究深入的揭示并拓宽了m6A甲基化研究进展及放疗抵抗的调控机制，为以后探索非小细胞肺癌放疗抵抗提供了靶点和预后标志物。