Integrative genomics identifies candidate microRNAs for pathogenesis of experimental biliary atresia

Abstract

Background: Biliary atresia is a fibroinflammatory obstruction of extrahepatic bile duct that leads to end-stage liver disease in children. Despite advances in understanding the pathogenesis of biliary atresia, very little is known about the role of microRNAs (miRNAs) in onset and progression of the disease. In this study, we aimed to investigate the entire biliary transcriptome to identify miRNAs with potential role in the pathogenesis of bile duct obstruction.

Results: By profiling the expression levels of miRNA in extrahepatic bile ducts and gallbladder (EHBDs) from a murine model of biliary atresia, we identified 14 miRNAs whose expression was suppressed at the times of duct obstruction and atresia (≥2 fold suppression, P < 0.05, FDR 5%). Next, we obtained 2,216 putative target genes of the 14 miRNAs using in silico target prediction algorithms. By integrating this result with a genome-wide gene expression analysis of the same tissue (≥2 fold increase, P < 0.05, FDR 5%), we identified 26 potential target genes with coordinate expression by the 14 miRNAs. Functional analysis of these target genes revealed a significant relevance of miR-30b/c, -133a/b, -195, -200a, -320 and -365 based on increases in expression of at least 3 target genes in the same tissue and 1st-to-3rd tier links with genes and gene-groups regulating organogenesis and immune response. These miRNAs showed higher expression in EHBDs above livers, a unique expression in cholangiocytes and the subepithelial compartment, and were downregulated in a cholangiocyte cell line after RRV infection.

Conclusions: Integrative genomics reveals functional relevance of miR-30b/c, -133a/b, -195, -200a, -320 and -365. The coordinate expression of miRNAs and target genes in a temporal-spatial fashion suggests a regulatory role of these miRNAs in pathogenesis of experimental biliary atresia.

Figure 1

Selection of miRNAs based on decreased expression at days 7 and 14 after RRV challenge. One-way cluster analysis depicts the changes in expression levels of 14 miRNAs in EHBDs at 3, 7 and 14 days after RRV challenge relative to normal saline (NS) controls. Each column represents expression levels from pooled samples of 2–6 EHBDs. Expression levels are depicted as color variation from low (blue) to high (red).

Figure 2

Expression levels of potential target genes in EHBDs after RRV challenge. One-way cluster analyses depict the changes in expression levels of mRNAs at 3, 7, and 14 days after RRV challenge relative to normal saline (NS) controls. Panel A shows a profile based on the differential expression at 7 days, while Panel B at 14 days. Each column represents expression levels from pooled samples of 2–6 EHBDs. RRV in red colored fonts indicate the time point used to select gene groups based on a significant level of expression in RRV group relative to NS controls.

Figure 3

Combined miRNA/mRNA regulatory and functional enrichment network. The network for miRNAs and their potential targets at day 7 after RRV challenge (listed in Table 3) was drawn based on the results of functional enrichment analysis performed using ToppCluster. 13 biological processes (light brown squares) and 2 pathways (blue diamonds) were overrepresented by 8 miRNAs (yellow circles) with their 14 potential targets (green hexagons). M: Biological processes related to morphology; I: Biological processes and a pathway related to Inflammation; L: A pathway related to Leukemia.

Figure 4

Expression of miRNAs in the liver and extrahepatic bile ducts (EHBDs). Expression levels in the liver and EHBDs of the same miRNAs depicted in Figure 4 during postnatal development. Expression of miRNAs was normalized against U6 miRNA, followed by fold change calculations using expression levels of adult livers. *P < 0.05, **P < 0.01, ***P < 0.001. Values are expressed as mean ± SEM.

Figure 5

Cellular localization of the miRNAs in EHBDs. In situ hybridization performed on EHBDs from 7-day old neonatal mice reveals substantial expression of the miRNAs in cholangiocytes (arrows) and stromal cells in the subepithelial compartment (arrowheads). Probe for U6 microRNA served as positive control and scramble probe served as negative control. Asterisks indicate the bile duct lumen. Scale bar: 50 μm.

Figure 6

Expression of miRNAs after RRV infection in a cultured cholangiocyte cell line. Expression levels of the 6 miRNAs in Figure 5 were quantified 24 hours after infection with RRV at MOI of 100. Expression of miR-365, miR-195, miR-30c and miR-200a were significantly downregulated in RRV infected cholangiocytes. Expression of miRNAs was normalized against U6 miRNA, *P < 0.05, **P < 0.01. Values are expressed as mean ± SEM.