Functional characters of non-coding RNAs in alcoholic liver injury

酒精性肝病(ALD)是美国最常见的慢性肝损伤形式之一。 长期饮用酒精会导致肝脏中的有毒代谢物，并加剧内皮细胞的衰老 以及导致炎症和肝脏损伤的功能障碍。衰老与疾病的严重程度和贫乏有关 包括酒精性肝病在内的各种肝病的预后及其相关的渐进性改变 肝组织和细胞包括肝窦内皮细胞的结构和功能。内皮细胞 功能障碍是肝病发生发展的早期病理生理标志。衰老，衰老 细胞老化的等价物，被认为与内皮功能障碍有关。事实上，增强了 细胞衰老与酒精相关性脂肪性肝病的不良结局有关，而 与肝脏/内皮细胞相关的肝脏衰老介质p21和肿瘤坏死因子-α增加 酒精性肝病肝硬变的功能障碍。越来越多的证据支持非编码RNA(NcRNAs)在 通过调节基因表达，在多种细胞途径中发挥作用。事实上，两者之间存在着很强的联系 乙醇代谢与内毒素/脂多糖诱导的细胞衰老和内皮 ALDS的功能障碍。直接与门脉循环相连的肝窦是第一道屏障。 对抗这些炎性和有害的刺激。我们有新的初步数据显示有选择性 NcRNA/microRNA基因在ALD动物的肝脏标本中异常表达并受到调控 并参与乙醇新陈代谢。特别是，我们发现：(A)胃内乙醇喂养 显著增加细胞衰老启动子Egr1、PAI-1和Id1的表达并使其沉默 P53效应分子E2F1和IGFBP3；(B)乙醇促进microRNA-34a和P53的表达并改变 它们的靶基因SIRT1、PPARα和HNF4α导致肝脏的衰老表型 (C)抗miR-34a/p53/TLR4抗体对miR-34a/p53/TLR4的抑制作用 CRISPR/Cas9方法成功地恢复了乙醇处理的小鼠的酒精性肝损伤 活着。尽管综合证据支持ncRNA与酒精性肝病之间的联系，但有 是确定乙醇依赖的miRNAs促进 酒精性肝损伤。我们的长期目标是确定导致酒精的潜在机制- 使诱发肝病有新的机理基础，临床有效的预防或治疗策略 可以被开发出来。这项提议的目标是确定乙醇依赖的ncRNAs如何调节 内皮功能障碍和衰老在酒精性肝病进展中的作用。我们的中央 假说认为酒精依赖的miRNAs通过调节 内皮细胞中的细胞衰老和功能障碍。这一假说是基于 现有文献和我们自己的初步数据如上所述。以下三个具体目标是 建议：首先，我们将确定ncRNA介导的细胞衰老和内皮细胞的作用 酒精性肝损伤加速衰老小鼠体内功能障碍(目标1)。第二，我们将 确定P53-miR-34a轴下游参与内皮细胞衰老和 体内miR-34a和p53基因敲除小鼠的功能障碍(目标2)。此外，我们将确定 内皮祖细胞相关的ncRNA富集胞外囊泡的抗衰老/抗衰老作用 体内酒精性肝损伤的内皮功能障碍和恢复(目标3)。在完成 提出的研究，是我们期待产生重要的积极影响，因为一个机械论 了解ncRNAs在酒精性损伤、内皮细胞和衰老中的作用 肝脏疾病可能为循证临床有用的发展提供基础 治疗或预防人类酒精性肝病的方法，包括美国退伍军人。

Alcoholic liver disease (ALD) is one of the most common forms of chronic liver injury in the United States. Chronic ethanol consumption results in toxic metabolites in the liver and increases endothelial senescence and dysfunction leading to inflammation and liver damage. Aging is linked with the severity and poor prognosis of various liver diseases including alcoholic liver diseases and is associated gradual alteration of structure and function in liver tissues and cells including liver sinusoidal endothelial cells. Endothelial dysfunction is an early pathophysiological hallmark in the development of liver disease. Senescence, the cellular equivalent of aging, was proposed to be involved in endothelial dysfunction. Indeed, enhanced cellular senescence was associated with a poor outcome in alcohol-related fatty liver disease, while increased hepatic senescence mediators such as p21 and TNF-α that were related to liver/endothelial dysfunction in ALD cirrhosis. Increasing evidence supports that non-coding RNAs (ncRNAs) play a central role in various cellular pathways by regulating gene expression. Indeed, there is a strong link between ethanol metabolism and endotoxin/lipopolysaccharide (LPS) induced cellular senescence and endothelial dysfunction in ALDs. Hepatic sinusoids, connected directly to the portal circulation, serve as the first barrier against these inflammatory and noxious stimuli. We have novel preliminary data showing that selective ncRNA/microRNA genes are aberrantly expressed in liver specimens from ALD animals and are regulated and involved in ethanol metabolism. In particular, we have found: (a) intra-gastric ethanol feeding significantly increased the expression of cellular senescence initiators EGR1, PAI-1 and Id1 and silenced p53 effectors E2F1 and IGFBP3; (b) ethanol enhanced microRNA-34a and p53 expressions and altered their target genes such as SIRT1, PPARα and HNF4α, which leads to senescence phenotypes in liver sinusoidal endothelial cells (LSECs); (c) Inhibition of miR-34a/p53/TLR4 by anti-miR-34a/p53 Morpholino and CRISPR/cas9 approaches has successfully recovered alcoholic liver injury in ethanol treated mice in vivo. Although the combined evidence supports a link between ncRNAs and alcoholic liver disease, there is a critical need to determine the underlying mechanism whereby ethanol-dependent miRNAs promote alcoholic liver injury. Our long-term goal is to determine underlying mechanisms contributing to alcohol- induced liver disease so that new mechanism-based, clinically effective prevention or treatment strategies can be developed. The objective for this proposal is to determine how ethanol- dependent ncRNAs mediate endothelial dysfunction and senescence in the progression of alcoholic liver diseases. Our central hypothesis is that ethanol-dependent miRNAs contribute to alcoholic liver injury through regulation of cellular senescence and dysfunction in endothelial cells. This hypothesis was formulated based upon the existing literature and our own preliminary data described above. The following three specific aims are proposed: First, we will determine the effects of ncRNA mediated cellular senescence and endothelial dysfunction in senescence accelerated mice with alcoholic liver injury in vivo (Aim 1). Second, we will identify the downstream targets of p53-miR-34a axis that are involved in endothelial senescence and dysfunction in miR-34a and p53 knockout mice in vivo (Aim 2). Further, we will determine the effects of endothelial progenitor cell related ncRNAs enriched extracellular vesicles on anti-senescence/anti- endothelial dysfunction and recovery of alcoholic liver injury in vivo (Aim 3). At the completion of the proposed research, it is our expectation to have an important positive impact because a mechanistic understanding of the role ncRNAs play in alcohol related injury, endothelial cell and senescence mediated liver disease is likely to provide a foundation for the development of evidence-based clinically useful approaches to treat or prevent human alcoholic liver diseases including US veterans.