m6A Epitranscriptomics in Toxicant Associated Steatohepatitis

项目摘要/摘要 暴露在包括多氯联苯在内的持久性环境污染物中会导致 代谢性疾病包括毒物相关性脂肪性肝炎(TASH)，这是一种非酒精性脂肪肝 疾病(NAFLD)。多氯联苯与TASH、肝细胞癌、肝脏改变呈正相关 酶，以及人类队列中的死亡率。完全了解多氯联苯的作用机理 第一次“热门”与高脂肪的“西方饮食”(HFD)相结合作为第二次“热门”，以增加甘油三酯的积累。 肝纤维化和炎症(TASH的特征)仍然难以捉摸。此外，尽管肝脏具有性行为 多氯联苯暴露后，人类健康结局中的二型性和性别差异是公认的 尽管已有报道，但很少有针对这些差异的机械性研究。N(6)-甲基腺苷(M6A)是 转录的RNA最常见的动态修饰。M6A在肝脏中的调节及其作用 表位转录和多氯联苯暴露的影响尚不清楚。此应用程序通过以下方式解决RFA-ES-19-002 研究改变的m6A及其编写器、读取器和擦除器在体内TASH中的作用，使用已建立的 印刷电路板曝光模型。初步RNA-SEQ数据显示，接触HFD Aroclor 1260(多氯联苯)男性肝脏 C57BL/6J小鼠m6A甲基转移酶复合体蛋白Rbm15的转录水平降低并改变 M6A读取器Ythdf1和Ythdc1/2的水平。这些HFD Aroclor 1260诱导的变化是否也 以及它们如何影响m6A表位转录组和肝细胞动态平衡或肝功能 未知。我们最近报告了多氯联苯引起的肝脏炎症和脂代谢改变的增加。 雌鼠对雄鼠。这项探索性研究的目标是确定多氯联苯/-HFD暴露对 雄性和雌性小鼠肝脏m6A表位基因的活体表达。我们将测试改变了m6A的假设 特定转录本的水平在体内的TASH中发挥作用。综合分析将确定m6A表位转录组- 调节TASH病理生理的信号通路的介导变化。这一目标将在1)之前实现 HFD Aroclor 1260(非二恶英混合物)中m6A介导的转录组改变的鉴定 最能模拟多氯联苯在人体脂肪组织中生物积累的多氯联苯亚型)，以及HFD Aroclor 1260- 暴露雄性和雌性小鼠肝脏；2)确定HFD、Aroclor 1260或HFD Aroclor 1260是否暴露 改变m6A的编写者、读取者和擦除者在小鼠肝脏中的表达。综合分析将确定 M6A表位转录组介导的调节TASH病理生理的信号通路变化可能是 治疗干预的靶点。在未来，敲除或过度表达在这个基因中发现的关键蛋白 研究将证实它们在观察到的M6A变化和下游信号转导中的作用。

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.