N6甲基腺嘌呤（m6A）甲基化介导miRNA21表达在环境镉暴露致肾损伤中的作用

Cadmium (Cd) is the most serious inorganic pollutant present in soil in China. Cd exposure can activate the stress response in body, and result in renal injury. Recent studies reported that the expression change of RNA methylation modification enzymes will induce the methylation of RNA (N6-methyladenosine, m6A) under stress condition. M6A will promote the initiation of pre-miRNA biogenesis. Our previous studies suggested that the expression of miRNA21 was significantly up-regulated after Cd exposure, and was related to renal dysfunction. However, whether or not renal injury induced by Cd exposure is related to m6A methylation, and whether or not differential expression of the methylation modification enzymes contribute to regulate the expression of miRNA21 then alter downstream target gene expression and induce the renal injury, remain incompletely understood. Based on the comprehensive evaluation of exposure and renal dysfunction of the environmental exposure to Cd, we will carry out the studies of RNA m6A methylation profiles in peripheral blood use the MeRIP-seq technology in different population exposed to different levels of Cd. Screen the differential methylation enzymes and validate them. The RNAi technology will be used to evaluate the regulative role of methylation enzymes on miRNA21 in vitro. The research project will explore the correlation between Cd-RNA methylation-miRNA21-renal injury, elucidate the mechanism of renal injury and look for earlier biomarker of Cd exposure.

镉是我国土壤中的首要无机污染物，能诱导机体的应激反应，造成肾损伤。近期研究发现应激状态下RNA甲基化修饰酶表达改变能够诱导RNA -N6甲基腺嘌呤（m6A）甲基化，进一步调控miRNA前体的生成。前期的研究结果显示镉暴露能引起miRNA21表达上调，并与肾损伤关系密切。但镉暴露是否能够诱导RNAm6A修饰？镉是否通过诱导甲基化修饰酶差异表达影响miRNA21表达，影响下游基因表达导致肾损伤？尚未见相关报道。本研究拟在对人群镉暴露水平和肾损伤进行综合评估的基础上，应用m6A抗体特异结合免疫沉淀的测序方法（MeRIP-seq），构建外周血全基因组RNAm6A甲基化修饰谱，筛查并验证差异表达的m6A修饰酶；利用RNA干扰技术体外研究修饰酶对miRNA21的调控作用及靶基因表达的影响，探讨镉-m6A甲基化-miRNA21-肾损伤之间的相关性，为阐明镉致肾损伤的机制以及寻找早期标志提供依据。

慢性肾脏疾病（CKD）的患病率不断增长，已经成为全球重要的公共健康问题，但有关CKD的影响因素和致病机制仍不明确。镉由于其生物半减期长，主要在肾脏蓄积，对肾脏的毒性作用受到广泛关注。本研究采用MeRIP-seq构建镉暴露人群外周血全基因组RNAm6A甲基化修饰谱，筛查并验证差异表达的m6A修饰酶。获得以下研究结果：（1）肾功能异常组研究对象尿镉水平高于对照组，且UNAG水平随尿镉水平的升高而升高，肾功能异常组去甲基化转移酶FTO mRNA表达水平与对照组比较差异具有统计学意义。经Nomogram列线图预测模型发现尿镉为肾功能异常的危险因素，FTO为肾功能的保护因素。MeRIP-seq分析发现肾功能异常且尿镉水平>15μg/gCr和肾功能正常组且尿镉＜5μg/gCr比较，发现280个差异表达基因，其中与m6A甲基化差异Peak相关的有13个基因。KEGG富集分析差异表达基因主要参与肾细胞癌、RAS信号通路、RNA降解、生物素代谢、Toll样受体信号通路、前列腺癌等过程；GO分析生物过程主要参与信号传导过程、RNA聚合酶Ⅱ对转录的正调控、多细胞生物发育、氧化还原过程、脂质代谢过程和磷酸化过程等。（2）体外实验研究结果显示CdCl2染毒肾小管上皮细胞（HKC）对细胞增殖、细胞周期和细胞凋亡以及细胞的间充质转化等生物学特征均有影响，且导致pri-miRNA21表达下调， miRNA21、METTL3、METTL14、和FTO表达均上调；进一步进行MeRIP-seq分析，对差异甲基化基因富集分析结果显示差异m6A peak与TGF-β、MAPK等信号通道有关。基于人群和体外实验研究，均显示FTO存在差异表达，进一步通过慢病毒转染构建miRNA21低表达细胞系，给予FTO抑制剂FB23-2后，未发现镉染毒对细胞TGF-β1表达的影响，也未发现m6A甲基化酶对镉染毒HKC细胞的调控作用。由此我们揭示了RNAm6A在镉暴露致肾功能损伤中发挥一定的作用，从而为镉致肾功能损伤机制研究提供了新视角。