m6A识别蛋白YTHDF2调控UBXN1/NF-κB信号轴参与恶性进展胶质瘤化疗抵抗的机制研究

Activation of NF-kB signaling is important for the chemotherapy-resistance during malignant progression of glioma. However, the underlying mechanism for regulating the activation of NF-kB signaling has not been fully elucidated. Our recent study indicated that m6A modification is highly correlated with the activation of the NF-kB signaling pathway and chemotherapy resistance in glioma. Moreover, our preliminary data showing that knockdown the expression of YTHDF2, a recognition protein for m6A modification, could increase the sensitivity of glioma cells to chemotherapy, suppress the activation of NF-kB signaling pathway and up-regulated the expression of NF-kB signaling inhibitor UBXN1. In addition, we also confirmed that there was m6A modification on the mRNA of NF-kB signaling inhibitor UBXN1. Given that YTHDF2 can promote the degradation of target RNA by recognizing m6A, these findings suggested that YTHDF2 could regulate the UBXN1/NF-kB signal axis through the m6A modification on UBXN1 mRNA, and thus involved in the malignant progression and chemotherapy-resistance of gliomas. In this project, we will further dissect the following issues: (1) The effect of YTHDF2 expression level changes on UBXN1/NF-kB signal axis, and identifying the exact m6A modification site which mediates the interaction of YTHDF2 protein and UBXN1 mRNA; (2)The effect of YTHDF2 expression level changes on the cellular functions and chemotherapy sensitivity of glioma cells, and the roles of UBXN1/NF-kB signal axis in this process; (3) The effect of YTHDF2 expression level changes on the glioma growth rate, survival and chemotherapy sensitivity of mice with glioma, and UBXN1/NF-kB signal axis in in vivo model. This project will reveal the regulator mechanism of NF-kB signaling pathway in the perspective of m6A modification, and thus provide the theoretical basis and reference for improving the diagnosis and therapy of glioma at the level of RNA epigenetics.

NF-κB信号通路激活是恶性进展胶质瘤化疗抵抗的重要原因，其上游调控机制尚未完全阐明。申请人近期研究发现m6A修饰与胶质瘤NF-κB信号通路激活和化疗抵抗密切相关；敲低m6A识别蛋白YTHDF2可抑制NF-κB信号激活和胶质瘤细胞的化疗抵抗；NF-κB抑制因子UBXN1 mRNA存在m6A修饰。鉴于YTHDF2可通过识别m6A促使目标RNA降解，提示m6A修饰介导的YTHDF2对UBXN1/NF-κB信号轴（目标信号轴）的调控是胶质瘤化疗抵抗的潜在机制。本项目将研究：①介导YTHDF2促使UBXN1 mRNA降解的m6A修饰位点；② YTHDF2通过调控目标信号轴对胶质瘤细胞化疗敏感性及功能影响；③在体水平YTHDF2对目标信号轴、肿瘤化疗敏感性及生长的影响。旨在从RNA m6A修饰层面揭示NF-κB信号参与胶质瘤化疗抵抗的分子调控机制，为克服恶性进展胶质瘤化疗抵抗提供新思路和理论依据。

当前弥漫性胶质瘤的预后仍然很差，其恶性进展和替莫唑胺(TMZ)耐药性的机制尚不清楚。在本项目中，我们旨在阐明RNA N6,2 ' - o -二甲基腺苷(m6A)阅读器，YTH N6-甲基腺苷RNA结合蛋白2 (YTHDF2)在调节胶质瘤恶性进展和TMZ耐药中的作用和机制。我们使用了几个独立的临床数据集评估了YTHDF2 mRNA在不同胶质瘤亚型中的表达水平和潜在生物学功能。我们通过western blotting、定量聚合酶链式反应和免疫组化评价YTHDF2等分子在人、小鼠肿瘤组织、以及细胞中的表达水平。利用病毒感染敲低和过表达靶分子，然后评估YTHDF2、甲基转移酶样3 (METTL3)和UBX结构域蛋白1 (UBXN1)在细胞和原位异种移植模型中对胶质瘤恶性肿瘤和TMZ耐药的影响。通过RNA免疫沉淀(RIP)、甲基化RIP和RNA稳定性实验，研究YTHDF2和METTL3的致癌和耐药的机制。我们的研究结果显示YTHDF2的表达与胶质瘤的恶性程度较高的分子亚型以及预后较差呈正相关。在体外和体内实验中，YTHDF2均能促进胶质瘤恶性进展。敲除YTHDF2也可增加胶质瘤细胞对TMZ的敏感性。机制上，YTHDF2通过METTL3介导的m6A加速UBXN1 mRNA降解，进而促进NF-κB激活。我们进一步发现UBXN1过表达减弱了YTHDF2过表达的致癌作用，并与YTHDF2表达升高的患者的生存率有关。总之，我们的研究结果证实了YTHDF2促进胶质瘤的恶性进展和TMZ耐药，并在在m6A修饰层面揭示了UBXN1激活NF-κB的上游调控机制，本研究过程中建立的细胞模型和一些胶质瘤耐药细胞株可为进一步开发胶质瘤分子检测工具提供基础。