The MicroRNA Network and the Deregulation of Liver Regeneration by Ethanol

描述（申请人提供）：肝脏再生是临床重要的组织修复机制，通过肝细胞的完全分化增殖促进急性损伤后肝脏肿块的恢复。慢性乙醇摄入干扰肝脏再生过程，这可能导致酒精性肝病的进展。我们和其他人发现，慢性乙醇喂养改变了大鼠部分肝切除术（PHx）后残余肝脏中发生的microRNA变化模式。值得注意的是，我们的研究表明，在肝细胞增殖受到严重抑制的情况下，乙醇喂养的大鼠的残肝中，一种假定的促增殖microRNA物种miR-21增加了。此外，通过给予miR-21 （AM21）的锁定核酸（LNA）拮抗剂抑制miR-21可以克服PHx后乙醇诱导的肝细胞增殖抑制，这表明miR-21有助于乙醇依赖性的再生抑制。残肝的基因表达谱分析显示，PHX后乙醇喂养的大鼠中，一簇促纤维化肝星状细胞（HSC）标记物上调，但在am21处理的动物中，这一簇标记物的上调受到抑制，这表明HSC的激活可能在乙醇抑制肝脏再生中发挥作用。有趣的是，AM21对miR-21的抑制导致了一组其他microrna的同时上调，这表明一个调节microrna网络参与了乙醇处理动物的再生恢复。我们建议分析再生肝脏中这种调控microRNA网络所针对的细胞类型特异性过程。提出以下具体目的：1)评估miR-21及其相互作用的miRNA调控网络作为细胞类型特异性介质对乙醇对PHx再生反应的抑制作用。这将涉及通过HITS-CLIP分析识别miR-21靶点，并分析通过激光捕获显微解剖（LCM）从残余组织中获得的实质细胞、星状细胞和其他细胞类型的群体，以了解microRNA的变化和相应的基因表达变化；2)评估与miR-21平行或反平行变化的microRNA物种在am21处理动物对PHx的反应中的网络作用，并评估它们在乙醇抑制细胞增殖后恢复中的作用。这些探索性研究将指导未来深入分析酒精诱导的肝脏再生中microRNA网络解除管制的性质，并具有确定酒精性肝病治疗新靶点的长期潜力。

DESCRIPTION (provided by applicant): Liver regeneration is a clinically important tissue repair mechanism that facilitates the recovery of liver mass after acute damage through proliferation of fully differentiated hepatocytes. Chronic ethanol intake interferes with the process of liver regeneration, which may contribute to the progression of alcoholic liver disease. We and others have found that chronic ethanol feeding alters the pattern of microRNA changes occurring in the remnant liver after partial hepatectomy (PHx) in the rat. Remarkably, our studies demonstrated that a presumed pro-proliferative microRNA species, miR-21, was increased in the remnant liver in ethanol-fed rats under conditions where hepatocyte proliferation was severely inhibited. Moreover, inhibition of miR-21 by the administration of a locked nucleic acid (LNA) antagonist of miR-21 (AM21) could overcome the ethanol- induced suppression of liver cell proliferation after PHx, suggesting that miR-21 contributes to ethanol- dependent inhibition of regeneration. A gene expression profiling analysis of the remnant liver demonstrated that a cluster of pro-fibrogenic hepatic stellate cell (HSC) markers was upregulated in ethanol-fed rats after PHX, but the upregulation of this cluster was suppressed in AM21-treated animals, indicating that HSC activation may play a role in the ethanol suppression of liver regeneration. Interestingly, inhibition of miR-21 by AM21 caused a concomitant upregulation of a cluster of other microRNAs, suggesting the involvement of a network of regulatory microRNAs mediating the recovery of regeneration in ethanol-treated animals. We propose to analyze the cell-type specific processes in the regenerating liver that are targeted by this regulatory microRNA network. The following specific aims are proposed: 1) To evaluate the role of miR-21 and its interacting miRNA regulatory network as cell type specific mediators of the suppressive effect of ethanol on the regenerative response to PHx. This will involve identifying miR-21 targets by HITS-CLIP analysis and analyzing populations of parenchymal cells, stellate cells and other cell types obtained from the remnant tissue by laser capture microdissection (LCM) for microRNA changes and corresponding changes in gene expression; 2) To evaluate the network roles of microRNA species that show parallel or anti-parallel changes relative to miR-21 in the response to PHx in AM21-treated animals and assessing their role in the recovery from ethanol inhibition of cell proliferation. These exploratory studies will guide a future in-depth analyses of the natur of the alcohol-induced deregulation of the microRNA network in liver regeneration with the long-term potential of identifying novel targets for therapy of alcoholic liver disease.