MicroRNAs as Mediators in the Pathogenesis of Non-Alcoholic Fatty Liver Disease and Steatohepatitis

PP Becker; B Niesler; O Tschopp; F Berr; A Canbay; T Dandekar; B Müllhaupt; J Schmitt; A Geier

doi:10.1055/s-0033-1360871

Zeitschrift für Gastroenterologie, Inhaltsverzeichnis

MicroRNAs as Mediators in the Pathogenesis of Non-Alcoholic Fatty Liver Disease and Steatohepatitis

PP Becker ¹, B Niesler ³, O Tschopp ⁴, F Berr ⁸, A Canbay ⁷, T Dandekar ⁵, B Müllhaupt ⁴, J Schmitt ², A Geier ²

¹University Hospital Zürich (USZ), Department of Gastroenterology and Hepatology, Zürich, Switzerland
²University Hospital Würzburg (UKW), Division of Hepatology, Würzburg, Germany
³Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany
⁴University Hospital Zürich (USZ), Department of Endocrinology, Diabetes and Clinical Nutrition, Zürich, Switzerland
⁵University of Würzburg, Bioinformatik, Biozentrum, Würzburg, Germany
⁶University of Zürich (UZH), Zürich Center for Integrative Human Physiology (ZIHP), Zürich, Switzerland
⁷University Hospital, University Duisburg-Essen, Department for Gastroenterology and Hepatology, Essen, Germany
⁸Paracelsus Medical University/Salzburger Landeskliniken (SALK), Department of Medicine I, Salzburg, Austria

Abstract

Objectives: The biological mechanisms of non-alcoholic fatty liver disease (NALFD) and non-alcoholic steatohepatitis (NASH) are not entirely understood particularly in human liver. Since microRNAs (miRNAs) have been discovered, scientists proofed a regulative function in various gene expression pathways.

Aim of this project is to study miRNAs expression and related target gene mRNA expression patterns in different stages of human fatty liver disease.

Methods: In an initial miRNA expression assay total RNA from liver tissue of 19 patients with NAFLD/NASH from the local biobank and five healthy controls was analyzed. A detailed metabolic and histological characterization was performed to obtain homogeneous groups for comparison of NAFLD vs. NASH. The NanoString® assay contains the measurement of 800 miRNAs in a multiplexed assay without previous amplification. Normalized data was analyzed. Newly described miRNA candidates as well as those already associated with fatty liver disease were replicated by quantitative rtPCR. Further replication of potential miRNA candidates was performed in a second cohort consisting of 35 NAFLD/NASH patients. At the same time a human gene expression array (Affymetrix PrimeView) was performed to investigate mRNA expression changes in miRNA target genes and involved signaling cascades in the initial cohort. Finally, expression changes and molecular interactions between miRNA and target genes were analyzed by mathematical network modeling.

Results: Primary analysis showed significant p-values (p < 0.05) after correction for almost 250 miRNAs. A significant difference of the mean compared to control (log2 expression (± 0.8 to ± 3.264)) was defined to select potential candidates. Comparing the two different stages respective to signs of hepatic inflammation (NAFLD vs. NASH), a total number of 71 miRNAs with a significant difference (0.0001<= p>= 0.042) were found. 13 miRNAs showed a significant deregulation in fatty liver disease compared to controls (e.g. miR 223 – 3 p and mir21 – 5 p). These results could be reproduced via rtPCR. miRNAs previously associated with metabolic or inflammatory pathways were newly assigned to either NAFLD or NASH. Established reference miRNAs (e.g. miR-33b, 34a-5 p) could be confirmed and further subjected to a comparative analysis of microRNA and mRNA expression. Pathophysiological consequences on central metabolic and inflammatory signaling pathways involved in the pathogenesis of human NAFLD/NASH were obtained by mathematical network modeling.

Conclusions: Our study identifies new functionally relevant miRNAs in liver tissue as mediators of central signaling pathways and clinically relevant pathophysiological events in fatty liver disease. These data from liver tissue imply a role of certain miRNAs as potential future prognostic biomarkers in serum to monitor the progression of fatty liver disease from bland steatosis to steatohepatitis.