Identification of candidate microRNA biomarkers of mouse cholangiocyte injury by global miRNAome analysis

Drug-induced liver injury (DILI) remains an issue in clinical and drug development settings causing large financial and health issues. This is in part due to the poor specificity and sensitivity of current standard circulating liver enzyme biomarkers, and research is ongoing to characterize more suitable novel candidates. Despite accounting for a small proportion of total liver cells, biliary epithelial cholangiocytes are vital to hepatic homeostasis due to their role in exporting toxic bile acids. Up to 40% of DILI cases can involve the biliary tract, yet much of the research for novel biomarkers has focused on hepatocellular injury, with miR-122 especially showing promise. We aimed to investigate novel microRNA (miR) biomarkers for cholangiocyte DILI. Characterization of basal murine hepatocyte and cholangiocyte miR profiles showed distinct expressions, which led to selection of a panel of cholangiocyte-enriched biomarker candidates, miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p and miR-429-3p. This panel was then evaluated in an α-Naphthyl isocyanate (ANIT) CD-1 mouse in vivo model of biliary toxicity. Liver phenotype and serum enzymes indicated that ANIT initially caused biliary perturbation before later progressing into wider hepatic injury. At 24-h following ANIT administration, during the biliary damage phase, miR-200a-3p showed the most consistent elevations in serum as per liquid biopsy vs controls, indicating a large dynamic range. No meaningful changes in serum miR-122-5p were evident until 48-h post-dose, once hepatocellular damage was taking place. miR expression differences were incorporated into a miR ratio calculation previously reported in a clinical DILI cohort, and results were able to successfully distinguish cholestatic or hepatocellular patterns of injury. This novel panel of cholangiocyte-selective miR-200 family members should be further investigated in larger cholestatic DILI cohorts, with miR-200a-3p in particular showing potential as a sensitive circulating marker of biliary toxicity with serum increases that precede miR-122-5p and hepatocellular damage.