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.