FTO介导RNA m6A修饰调控克唑替尼肝毒性的机制研究

Crizotinib hepatotoxicity is an urgent clinical issue during the treatment of non-small cell lung cancer, and the mechanism of toxicity remains unclear. It has been reported that crizotinib-induced hepatotoxicity is through the process of apoptosis, and epigenetic modification plays an important role in hepatotoxicity. In our previous study, we found that the level of N6-methyladenosine (m6A) was significantly increased in liver mRNA of mice with crizotinib-induced liver injury, and the expression of m6A demethylase FTO was significantly down-regulated. Besides, we found that crozotinib could promote hepatocyte apoptosis in mice. It has been confirmed that knockdown of FTO can promote the apoptosis of L78 and other cells. The results of sequencing showed that the up-regulated differential genes modified by m6A were mainly enriched in apoptosis-related pathways. Gadd45g may be a target gene regulated by FTO. The role of m6A in hepatotoxicity induced by drug has not been studied so far. Based on these findings, we hypothesize that crizotinib increases the level of mRNA m6A modification of Gadd45g by inhibiting the expression of FTO, and up-regulate the expression of Gadd45g, which in turn results in hepatotoxicity. In this project, we intend to elucidate the mechanism of FTO-mediated m6A modification in regulating the hepatotoxicity of clozoltinib by means of molecular biology, epigenetics and bioinformatics at the cellular, animal model and clinical levels. It will provide new ideas for the prevention and treatment of hepatotoxicity induced by crizotinib.

克唑替尼肝毒性是非小细胞肺癌治疗中的临床难题，其机制亟待阐明。文献显示克唑替尼通过诱导肝细胞凋亡产生肝毒性，且表观遗传学修饰在肝毒性中有重要作用。我们预实验发现：克唑替尼肝损伤小鼠肝组织中mRNA N6-甲基腺嘌呤（m6A）水平显著升高，m6A去甲基化酶FTO显著下调，且肝细胞凋亡明显增加。文献报道，敲低FTO可促进L78等细胞凋亡。我们测序结果亦显示m6A上调的差异基因主要富集在凋亡通路，Gadd45g可能是FTO调控的靶基因。目前未见关于RNA m6A修饰调控克唑替尼肝毒性的报道。据此我们提出假说：克唑替尼可能通过抑制FTO表达，增加Gadd45g的mRNA m6A修饰水平，上调Gadd45g表达，诱导肝细胞凋亡导致肝毒性。本课题拟从细胞、动物及临床水平，利用分子生物学、表观组学和生物信息学等学科手段，阐明FTO介导m6A修饰调控克唑替尼肝毒性的机制，为克唑替尼肝毒性的防治提供新思路。

克唑替尼肝毒性是临床上亟待解决的难题，缺乏有效的防治手段，然而其机制仍不清楚，有待研究。文献显示表观遗传学修饰在肝毒性中有重要作用，m6A修饰是mRNA上最常见的一类表观修饰，在疾病发生中具有重要作用。本研究首先探讨了RNA m6A修饰与克唑替尼肝毒性关系，开展了克唑替尼在小鼠体内的组织分布研究。结果显示克唑替尼肝损伤小鼠肝组织mRNA m6A甲基化修饰水平显著升高；克唑替尼的主要靶器官是肺、肝和脾，少量的克唑替尼可以穿透血脑屏障。其次，采用代谢组和转录组联合分析解析了克唑替尼肝损伤小鼠的代谢特征和转录组变化，探讨其肝毒性机制。肝脏和血浆中均显著上调的差异代谢物是五羟色胺。对差异基因和差异代谢物进行相关性分析，发生显著变化的代谢物有：醇类和有机胺类、氨基酸及其衍生物、胆汁酸、苯和取代衍生物、脂肪酸、核苷酸及其代谢物等。最后，在动物和细胞水平的证实了克唑替尼可引起肝细胞凋亡，克唑替尼可使小鼠肝组织和肝细胞中m6A阅读蛋白YTHDF3显著下调，我们对小鼠肝组织进行m6A MeRIP-seq和RNA-Seq测序，结合生物信息学分析结果和功能实验数据筛选出m6A修饰调控克唑替尼肝毒性的靶基因Lcn2，并将阐明其调控的分子机制。本研究为防治克唑替尼肝毒性提供理论依据和基础，并为药物性肝损伤的表观遗传学研究提供新思路。