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Hepatic miRNA expression in experimental biliary atresia

ABSTRACT: Hepatic microRNA was measured by Exiqon array in the mouse RRV model of biliary atresia miRNA expression profiling. For the miRNA microarray experiment, RRV and saline injected animals were euthanized at 3, 8, and 14 days post infection (n=5 per treatment, per time point), and total RNA was purified from liver using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. A pooled common control was assembled from an equal RNA mass from each of the samples, and samples and control were separately dye-labeled, and hybridized to a miRcury v11.0 miRNA microarray (Exiqon) at the University of Pennsylvania School of Medicine Microarray core facility. Sample hybridization intensities were scored relative to the common control, and raw intensity data were normalized and analyzed using the SAM add-in for Microsoft Excel. MiRNAs exhibiting a fold-change of greater than 10% up or down, at a false discovery rate of 5% were chosen for further study.

ORGANISM(S): Mus musculus Rattus norvegicus Homo sapiens

PROVIDER: GSE33418 | GEO | 2011/11/04

SECONDARY ACCESSION(S): PRJNA148925

REPOSITORIES: GEO

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Project description:Biliary atresia (BA) is a rare cholestatic disease of unknown etiology that affects infants and shows an incidence of 1 out of 18,000 live births in Europe (1). The first therapeutic option is a timely performed portoenterostomy. However, the majority of patients suffer from a progressive inflammatory process, which leads to complete destruction of the extra- and intrahepatic biliary system followed by end-stage liver cirrhosis. Hence, BA is the leading indication for pediatric liver transplantation worldwide (2, 3). To understand the pathogenesis of the disease and improve theoutcome of BA patients, research has focused on the inflammatory process in liver and bile ducts, in which several factors are remarkably elevated, such as activated CD4 and CD8 T-cells, TNF alpha,IFN alpha and other proinflammatory TH1 cytokines (3-8). By the time of diagnosis, however, the disease has already reached an advanced state, characterized by the complete obstruction of the extrahepatic bile ducts with impaired bile flow and fibrosis or cirrhosis of the liver. Therefore, studies in humans focusing on the trigger mechanism of BA are limited due to the paucity of liver and availability of bile duct tissue for research. One infectious animal model has been developed, in which newborn Balb/c mice exclusively show the experimental BA phenotype after infection with rhesus rotavirus (RRV) (9, 10). This model allows the analysis of the inflammatory reactions in liver and bile ducts at early steps in the development of bile duct atresia (11-20). Furthermore, inbred mouse strains have been shown to have a different susceptibility for the development of experimental BA, suggesting that Balb/c mice have an immunological gap responsible for disease progression (10, 12). The aim of this study was to identify key genes responsible for the BA phenotype by comparing the transcriptomes at an early time point after virus infection, i.e. before bile duct atresia, between two mouse strains with different susceptibilities to BA. Differences in the virus titration and the clinical course of infected mice were analyzed, and variations in the hepatic gene response assessed by comparative microarray assays were correlated to variances in the hepatic inflammatory reaction. Balb/c mice and C57Black/6 (Black/6) mice were infected with RRV postpartum and signs of BA and survival were noted. Liver sections of diseased, healthy and control animals were assessed for T-cell expression, and the virus loads were determined. Second, mice were sacrificed after three days, and isolated hepatic RNA was subjected to gene expression analysis using Affymetrix Gene Chip MOE 430 2.0.We compared three individual expression profiles from RRV-infected Balb/c mice against 2 individual expression profiles from RRV-infected C57/BL6 control mice using the Affymetrix GeneChip MOE 430 2.0.

Project description:MicroRNAs (miRNAs) are an endogenous conserved class of non-coding 20–22 nt small RNAs that regulate gene expression at post-transcriptional level by mostly binding to 3′-UTR of target mRNAs, leading to mRNA degradation or translation inhibition. Recent reports demonstrate a role for miRNA expression in the disease progression and outcome. By now, many researcher focusing on miRNA expression profiles in Barrett's esophagus and esophageal adenocarcinoma have been reported. Nevertheless, there is still a little information available about specific miRNA expression pattern and their roles in ESCC. To develop novel diagnostic and therapeutic targets for esophageal squamous cancer, we first investigated the expression profile of miRNA in three pairs of ESCC clinical samples. Tissues of ESCC and the matched normal counterparts were obtained from surgical specimens immediately after resection from patients undergoing primary surgical treatment of esophageal carcinoma from the Department of Tumor Surgery of Shantou Central Hospital, China. RNA labeling and hybridization were completed by KangChen Bio-tech Inc. (Shanghai, China) according to the manufacturer's instructions. Briefly, total RNA from three pairs of esophageal carcinoma and matched normal tissues were isolated by Trizol (Invitrogen, USA) and purified by RNeasy mini kit (QIAGEN, German). The concentration and quality of total RNA were measured by NanoDrop ND-1000 at 260 and 280 nm (A260/280) and checked by gel electrophoresis. Each RNA sample from three pairs of ESCC was separately labeled either using the miRCURY Hy3/Hy5 labeling kit and hybridized on the six miRCURYTM locked nucleic acid (LNA) array version 11.0 (Exiqon, Denmark), which contains probes for 1700 mature miRNA. Scans were quantified by using GenePix software (Molecular Devices). The data were exported to Microsoft Excel worksheets, log2 transformed, normalized using global Lowess (Locally Weighted Scatter plot Smoothing) regression algorithm (MIDAS, TIGR Microarray Data Analysis System).

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Hepatic miRNA expression in experimental biliary atresia

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