Ethanol Effects on the Transcriptional Regulatory Network in Liver Regeneration

 描述(申请人提供)：肝脏再生是一个临床上重要的组织修复过程，在这个过程中，肝脏通过驱动分化的肝细胞的同步复制来对损伤做出反应。这一过程涉及来自不同细胞类型的精心编排的信号与系统性因素的相互作用，以恢复功能性组织块。慢性酒精摄入抑制了肝脏再生，这种受损的修复反应可能导致酒精性肝病的风险。再生反应的开始和发展以及乙醇对其放松调控的机制仍未得到很好的描述。在以前的研究中，我们对乙醇喂养的大鼠和对照组大鼠肝部分切除(PHX)后的再生反应进行了详细的基因表达和MicroRNA图谱研究，并开发了新的生物信息学工具来突出细胞类型的特定转录特征。这些研究表明，慢性乙醇治疗影响控制肝细胞启动和复制的非实质细胞(NPC)，并与肝星状细胞(HSC)激活信号上调有关。我们获得的证据表明miR-21可能是这些差异反应的驱动因素。用特定的miR-21拮抗剂AM21抑制大鼠体内的miR-21，可以恢复乙醇喂养的大鼠的肝再生，同时抑制不同的HSC激活模式。在目前的应用中，我们以这些发现为基础，使用激光捕获显微解剖(LCM)分析不同类型的细胞如何对综合组织反应做出贡献，并通过计算建模方法整合实验数据，以预测再生如何因适应慢性乙醇暴露而受到抑制，并被miR-21抑制所挽救。(1)我们将分析酒精喂养的大鼠和对照组大鼠的瞬时再生动力学，并描述AM21治疗的不同影响，以表征有助于乙醇介导的肝再生抑制的miR-21靶向过程，重点关注NPC对细胞周期进程的贡献。我们将进一步比较大鼠和小鼠的肝再生模型，以评估miR-21‘S与细胞周期调节因子的相互作用在对苯丙氨酸有不同时间反应的动物中有何不同。(2)我们将通过使用LCM获得的单个细胞分析乙醇喂养动物与对照组相比细胞类型特定的转录组和microRNA表达的变化，来检验这样的假设，即对酒精摄入的适应改变了肝脏中不同类型细胞的时间反应，解除了对整合组织反应的调控。(3)我们将应用细胞类型特异性相互作用在肝脏再生中的作用的计算模型来迭代地评估表型分布动态变化的后果。我们集成的实验和计算分析将对驱动细胞表型分布和细胞间相互作用的调控miRNA网络产生新的见解，以及这些网络中的干预措施可以对抗乙醇诱导的肝修复抑制的机制，并应用于广泛范围的肝病的翻译和临床干预I。

 DESCRIPTION (provided by applicant): Liver regeneration is a clinically important tissue repair process in which the liver responds to injury by driving a synchronized replication of differentiated liver cells. The process involves the interplay of carefully orchestrated signals frm different cell types integrated with systemic factors to recover functional tissue mass. Liver regeneration is inhibited by chronic ethanol consumption and this impaired repair response may contribute to the risk for alcoholic liver disease. The mechanisms responsible for the onset and progression of the regenerative response and its deregulation by ethanol remain poorly characterized. In prior studies we carried out a detailed gene expression and microRNA profiling study of the regeneration response after 70% partial hepatectomy (PHx) in ethanol-fed and control rats and developed novel bioinformatics tools to highlight cell- type specific transcriptomic signatures. These studies indicate that chronic ethanol treatment affects non- parenchymal cells (NPCs) that control hepatocyte priming and replication and is associated with upregulation of a hepatic stellate cell (HSC) activation signature. We obtained evidence that miR-21 may be a driver of these differential responses. Inhibition of miR-21 in the rat in vivo by treatment with AM21, a specific miR-21 antagonist, allowed recovery of liver regeneration in ethanol-fed rats while suppressing the differential HSC activation profile. In the current application we build on these findings to analyze how different cell types contribute to the integrated tissue response using laser capture microdissection (LCM) and integrating the experimental data through a computational modeling approach to predict how regeneration is inhibited by adaptation to chronic ethanol exposure and rescued by miR-21 inhibition. (1) We will analyze the temporal regeneration dynamics in ethanol-fed and control rats and profile the differential impact of AM21 treatment to characterize the miR-21 target processes that contribute to the ethanol-mediated inhibition of liver regeneration, with a focus on the contribution of NPCs to cell cycle progression. We will further compare rat and mouse models of liver regeneration to evaluate how miR-21's interactions with cell cycle regulators differs in animals that have a different temporal response to PHx. (2) We will test the hypothesis that the adaptation to ethanol consumption alters the temporal response of different cell types in the liver, deregulating the integrated tissue response, by analyzing cell type-specific changes in the transcriptome and microRNA expression in ethanol-fed animals compared to controls by analyzing individual cells obtained using LCM. (3) We will apply a computational model of the role of cell-type specific interactions in liver regeneration to iteratively evaluate the consequences of dynamic changes in phenotype distribution. Our integrated experimental and computational analysis will yield new insights into the regulatory miRNA network driving cell phenotype distributions and cell-cell interactions, and the mechanisms through which interventions in these networks can counter ethanol-induced suppression of liver repair, with applications to translational and clinical interventions i in a broad range of liver diseases.