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.
The primary goal of the project is to understand the microRNA based regulatory mechanisms that underlie the chronic alcohol mediated deficiencies in liver repair and regeneration. We will identify the regulatory interactions between microRNAs as well as identify their target genes by perturbing the levels of specific microRNA and combining data on microRNA and gene expression and their interaction from individual cell types in regenerating liver.
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