Exosomal miR-122 as a sensitive marker for methotrexate and acetaminophen induced hepatocellular injury  

In vitro models for liver disease suffer from the lack of well-established and sensitive  biomarkers of cellular damage. miRNAs (small non-coding RNAs) represent potential  biomarkers for liver injury that can be detected in body fluids and cell culture media. Here, we  utilized 3D-HepaRG cultures for the identification of miRNAs as potentially sensitive markers  of hepatocellular damage, with a specific focus on their exosomal release. The 3D-cell  cultures displayed liver-like properties (e.g. metabolic activity, urea and albumin production),  and responded to acetaminophen (APAP)-induced toxicity. We explored APAP- and  methotrexate (MTX)-dependent changes in intracellular and released levels of three miRNAs  species: miR-122-5p and miR-192-5p, both associated with hepatic damage, and miR-34a 5p, involved in apoptosis. APAP-treatment led to enhanced release of miR-122-5p, miR-192- 5p and miR-34a-5p. Exosomes could be efficiently isolated from APAP-treated 3D-HepaRG,  characterized, and used for miRNA quantification. Absolute quantification of miR-122-5p  corroborated the release of this miRNA by both treatments. Moreover, total extracellular and  exosomal-miR-122-5p release occurred at concentrations lower than those leading to MTX induced apoptosis/necrosis. In conclusion, our results demonstrate the suitability of 3D HepaRG combined with exosomal miRNA measurement for the detection of hepatocellular  damage in vitro. Specifically, exosomal-miR-122-5p is a sensitive marker of APAP- and  MTX-induced injury.