The Study Of Regulation Of Gene Expression During Liver Regeneration

The liver has a remarkable capacity to regenerate itself in response to signals asphysical, chemical, nutritional, vascular, or virus-induced liver injury. In fact, the mainstrategies employed in the study of liver regeneration during the decades are based onsingle signal molecular, transcription factors and its related signal pathways, mRNAprofiling, and proteomic studies. However, these conventional methods are limited in fullyuncovering the molecular mechanisms by which the liver regenerates itself. Thus, thepresent study aims to investigate the microRNA profiling and transcription factor activityprofiling during different phases of liver regeneration.SectionⅠ: A study on the role and mechanism of differentially expressed microRNAsIn recent years, non-coding small RNAs have been shown to play fundamental rolesin many diseases and biological processes. MicroRNAs (miRNAs) are a class of smallregulatory RNAs which regulate many biological processes, including proliferation,differentiation, apoptosis and metabolism. Some studies have strongly indicated thatmicroRNAs are involved in hepatocellular carcinogenesis and liver regeneration. In thisstudy, we explore the differentially expressed microRNAs during the early and late phasesof liver regeneration in rats and mice, by using microRNA array and quantitative real-timePCR (qRT-PCR). Our study shows that miR-376b, miR-494and miR-127weresignificantly down-regulated during the early phase; while miR-34a was strongly induced(>5-folds) during the late phase of liver regeneration. Then the role and mechanism ofmiR-34a, miR-376b and miR-494were studied in cellular model.Functional study of miR-34a. Here, we show that miR-34a is significantly inducedafter partial hepatectomy. Introduction of miR-34a in BRL-3A cells could significantlyinhibit cell proliferation, and down-regulate the expression of inhibin βB (INHBB) andMet. In BRL-3A cells, INHBB was identified as a direct target gene of miR-34a. Moreimportantly, INHBB siRNA could significantly repress cell proliferation. Also, thedecrease of INHBB and Met is observed in regenerating liver. In exploring the upstreamregulator of miR-34a, we confirmed that p53could bind to the promoter region of miR-34ain regenerating livers and contribute to the activation miR-34a expression.MiR-34a-mediated regulation of INHBB and Met may collectively contribute to the termination of LR. These findings also reveal that a certain pattern of miRNA-relatedsignal pathway could exist in the process of LR.Functional study miR-376b and miR-494. Both miR-376b and miR-494are locatedin the mouse distal12chromosome, at the DLK1-GTL2domain. Introduction of miR-376band miR-494mimics into BNL-CL2cells could significantly inhibit cell proliferation.Then we combined the predicted miRNA target gene with proteomic evaluation (stableisotope labeling by amino acids in cell culture, SILAC) to find potential target genes ofboth miRNAs in regenerating livers. More importantly, we proved that miR-376b andmiR-494mimics could significantly reduce the expression of their potential target genes oncellular model. Therefore, we conclude that both miRNAs may play a role in the regulationof liver regeneration priming through these target genes.SectionⅡ: Identification of transcription factor activity profiles and transcriptionalnetworks during liver regenerationSo far, comprehensive transcription factor (TF) activity profiles during LR have notyet been reported. Thus, information regarding the pattern of gene expression andregulation networks during the first hours following PHx is still limited. In the presentstudy, we employed an advanced mouse oligonucleotide array-based transcription factorassay (MOUSE OATFA) platform that assays some200TF activities simultaneously,representing about10%of the predicted murine TFs in the investigation of liverregeneration priming.17TFs with>1.5-fold change at2h post-hepatectomy have beenfound, as comparing to SH control group. Some of these TFs were not known to beinvolved in this process, including USF-1. We then combined the TF signatures withmRNA expression profiles to construct experimental transcriptional regulatory networks inregenerating livers. Moreover, we focused on the transcriptional network of USF1, using invivo TF-binding data generated by ChIP-on-chip analysis. Based on these integratedanalytic results from independent and complementary resources, we sketched a map oftranscriptional regulatory networks involving USF1regulating the priming phase of LR.Based on the above analysis, the present study focused on the miRNA profiling,differentially expressed miRNAs, TF activity profiling and differentially regulated TFs inthe liver regeneration process generated by70%partial hepatectomy in rats or mice. Ourinvestigation will provide a better understanding of the molecular mechanism by which liver regenerates itself after surgery. It will also provide additional information about themechanism and therapeutic strategies for liver diseases.