Primary biliary cirrhosis is associated with altered hepatic microRNA expression

Abstract

MicroRNAs (miRNAs) are small RNA molecules that negatively regulate protein coding gene expression and are thought to play a critical role in many biological processes. Aberrant levels of miRNAs have been associated with numerous diseases and cancers, and as such, miRNAs have gain much interests as diagnostic biomarkers, and as therapeutic targets. However, their role in autoimmunity is largely unknown. The aims of this study are to: (1) identify differentially expressed miRNAs in human primary biliary cirrhosis (PBC); (2) validate these independently; and (3) identify potential targets of differentially expressed miRNAs. We compared the expression of 377 miRNAs in explanted livers form subjects with PBC versus controls with normal liver histology. A total of 35 independent miRNAs were found to be differentially expressed in PBC (p < 0.001). Quantitative PCR was employed to validate down-regulation of microRNA-122a (miR-122a) and miR-26a and the increased expression of miR-328 and miR-299-5p. The predicted targets of these miRNAs are known to affect cell proliferation, apoptosis, inflammation, oxidative stress, and metabolism. Our data are the first to demonstrate that PBC is characterized by altered expression of hepatic miRNA; however additional studies are required to demonstrate a causal link between those miRNA and the development of PBC.

Figure 1. Heat map and hierarchical clustering

Individual miRNA expression values were calculated as described in Materials & Methods. Duplicates of log2-transformed values of each spot were averaged and imported into Cluster 3.0. Hierarchical clustering and self-organizing maps were computed using the parameters described in Materials & Methods. Dendrograms and log2-transformed miRNA expression values were visualized using Java Treeview. miRNAs are presented that were significantly differentially regulated using a maximum false discovery rate of 5%.

Figure 2. Validation of microarray results for selected miRNAs

Real time threshold cycle values for each miRNA primer/probe set were normalized with RNU-48 primer/probe pair and compared to a calibrator [28]. The final values were imported into the statistical software Prism (version 4) and analyzed with the Mann Whitney U test. The results of the RT-PCR validation study correlate with the results generated with the microarrays

Figure 3. Multiplicity and cooperativity in miRNA-target gene interactions

Visualization of down-regulated and up-regulated Cytoscape pathways Gene Ontology biological process pathways were generated with the BiNGO plug-in in Cytoscape. Relative gene expressions are depicted by the size and colors of the nodes. Red nodes depict processes that are targeted mainly by up-regulated genes while blue represents that of down-regulated genes. A) Overview of biological processes. B-I) Selected highlighted process pathways