m6A Epitranscriptomics in Toxicant Associated Steatohepatitis

项目概要/摘要 接触持久性环境污染物，包括多氯联苯， 代谢性疾病，包括毒物相关性脂肪性肝炎（TASH），一种非酒精性脂肪肝 非酒精性脂肪肝（NAFLD）。多氯联苯与TASH、肝细胞癌、肝脏改变呈正相关 酶和死亡率。全面了解多氯联苯作为 第一次“打击”与高脂肪“西方饮食”（HFD）组合作为第二次"打击"，以增加甘油三酯积累， 肝纤维化和炎症（TASH的标志）仍然难以捉摸。此外，虽然肝性 二型性是公认的，在接触多氯联苯后，人类健康结果的性别差异 尽管已有报道，但很少有针对这些差异的机制研究。N（6）-甲基腺苷（m6 A）是 转录RNA最常见的动态修饰。m6 A在肝脏中的调节及作用 epitranscriptomics和PCB接触的影响是未知的。本申请通过以下方式解决RFA-ES-19-002 研究改变的m6 A及其写入器、读取器和擦除器在体内TASH中的作用，使用已建立的 PCB暴露模型。初步RNA-seq数据显示，来自HFD + Aroclor 1260（PCB）暴露男性的肝脏 C57 BL/6 J小鼠具有降低的m6 A甲基转移酶复合蛋白Rbm 15的转录水平和改变的m6 A甲基转移酶复合蛋白Rbm 15的转录水平。 m6 A阅读器Ythdf 1和Ythdc 1/2的水平。这些HFD + Aroclor 1260诱导的变化是否也 发生在女性中，以及它们如何影响m6 A表位组和肝细胞稳态或肝功能， 未知我们最近报道了多氯联苯诱导的肝脏炎症增加和脂质代谢改变， 雌性小鼠与雄性小鼠。本探索性研究的目的是确定PCB +/- HFD暴露对 体内对雄性和雌性小鼠肝脏m6 A表位转录组的影响。我们将检验m6 A基因改变的假设 特异性转录物的水平在体内TASH中起作用。综合分析将确定m6 A表位转录组- 调节TASH病理生理学的信号通路介导的变化。这一目标将通过1） HFD，Aroclor 1260（非二恶英样（NDL）混合物）中m6 A介导的转录组变化的鉴定 最能模拟PCB在人体脂肪组织中生物累积的PCB亚型），以及HFD +多氯联苯1260 - 暴露的雄性和雌性小鼠肝脏; 2）确定HFD、Aroclor 1260或HFD + Aroclor 1260暴露 改变小鼠肝脏中m6 A的写入者、读取者和擦除者的表达。综合分析将确定 调节TASH病理生理学的信号通路中m6 A表转录组介导的变化可能是 治疗干预的目标。在未来，敲低或过表达的关键蛋白质确定在这一点上， 这项研究将证实它们在观察到的m6 A变化和下游信号传导中的作用。

Project Summary/Abstract Exposure to persistent environmental contaminants including polychlorinated biphenyls (PCBs) contributes to metabolic diseases including toxicant-associated steatohepatitis (TASH), a form of nonalcoholic fatty liver disease (NAFLD). PCBs have been positively associated with TASH, hepatocellular cancer, altered liver enzymes, and mortality in human cohorts. A complete understanding of the mechanisms by which PCBs act as a 1st ‘hit’ in combination with a high fat ‘Western diet’ (HFD) as a 2nd ‘hit’ to increase triglyceride accumulation, fibrosis, and inflammation in the liver (hallmarks of TASH) remains elusive. Further, although liver sexual dimorphism is well-established and sex-specific differences in human health outcomes after PCB exposure have been reported, there are few mechanistic studies addressing these differences. N(6)-methyladenosine (m6A) is the most common dynamic modification of transcribed RNAs. The regulation and role of m6A in liver epitranscriptomics and the impact of PCB exposure is unknown. This application addresses RFA-ES-19-002 by investigating the role of altered m6A and its writers, readers, and erasers in TASH in vivo, using an established PCB exposure model. Preliminary RNA-seq data show that liver from HFD + Aroclor 1260 (PCB) exposed male C57BL/6J mice have reduced transcript levels of the m6A methyltransferase complex protein Rbm15 and altered levels of the m6A readers Ythdf1 and Ythdc1/2. Whether these HFD + Aroclor 1260-induced changes are also occur in females and how they affect the m6A epitranscriptome and hepatocyte homeostasis or liver function is unknown. We recently reported increased PCB-induced hepatic inflammation and altered lipid metabolism in female vs. male mice. The goal of this exploratory study is to determine the impact of PCB +/- HFD exposure in vivo on the hepatic m6A epitranscriptome in male and female mice. We will test the hypothesis that altered m6A levels of specific transcripts play a role in TASH in vivo. Integrated analysis will identify m6A epitranscriptome- mediated changes in signaling pathways regulating TASH pathophysiology. This goal will be achieved by 1) Identification of m6A-mediated transcriptome changes in HFD, Aroclor 1260 (a mixture of non-dioxin-like (NDL) PCB subtypes that best mimics the PCB bioaccumulation in human adipose tissue), and HFD + Aroclor 1260 – exposed male and female mouse liver; 2) Determination if HFD, Aroclor 1260, or HFD + Aroclor 1260 exposure alter the expression of the writers, readers, and erasers of m6A in mouse liver. Integrated analysis will identify m6A epitranscriptome-mediated changes in signaling pathways regulating TASH pathophysiology that may be targets for therapeutic intervention. In the future, knockdown or overexpression of key proteins identified in this study will confirm their role in the observed m6A changes and in downstream signaling.