Abstract
Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) resulting in the formation of fibrous scars. In the clinic, liver biopsies are the standard diagnostic method despite the potential for clinical complications. miRNAs are single-stranded, non-coding RNAs that can be detected in tissues, body fluids and cultured cells. The regulation of many miRNAs has been linked to tissue damage, including liver fibrosis in patients, resulting in aberrant miRNA expression/release. Experimental evidence also suggests that miRNAs are regulated in a similar manner in vitro and could thus serve as translational in vitro–in vivo biomarkers. In this work, we set out to identify and characterize biomarkers for liver fibrosis that could be used in vitro and clinically for research and diagnostic purposes. We focused on miRNAs released from hepatic 3D cultures exposed to methotrexate (MTX), which causes fibrosis, and acetaminophen (APAP), an acute hepatotoxicant with no clinically relevant association to liver fibrosis. Using a 3D in vitro model, we corroborated compound-specific responses as we show MTX induced a fibrotic response, and APAP did not. Performing miRNA-seq of cell culture supernatants, we identified potential miRNA biomarkers (miR-199a-5p, miR-214-3p, niRNA-125a-5p and miR-99b-5p) that were associated with a fibrotic phenotype and not with hepatocellular damage alone. Moreover, transfection of HSC with miR-199a-5p led to decreased expression of caveolin-1 and increased α-SMA expression, suggesting its role in HSC activation. In conclusion, we propose that extracellular miR-214-3p, miR-99b-5p, miR-125a-5p and specifically miR-199a-5p could contribute towards a panel of miRNAs for identifying liver fibrosis and that miR-199a-5p, miR-214-3p and miR-99b-5p are promoters of HSC activation.
Highlights
Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM), which distorts hepatic architecture through the formation of fibrous scars
Expression of CYP3A4 was detected under basal conditions and was strongly induced in MTs treated with RIF (Figure 1A)
THP-1 and hTERT-HSC were identified by CD68 and vimentin staining, confirming successful incorporation of all three cells lines, as previously reported by Prestigiacomo et al [49] TGF-β1 is a key cytokine in liver fibrosis progression and is described as a key event (KE) in the adverse outcome pathway (AOP) for liver fibrosis
Summary
Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM), which distorts hepatic architecture through the formation of fibrous scars. A leading cause of liver damage involves the intake of drugs, known as drug-induced liver injury (DILI), which is a major challenge in clinical medicine and drug development [8]. Certain drugs such as methotrexate (MTX) elicit chronic DILI in some patients and can lead to hepatic fibrosis and cirrhosis [9,10]. Other drugs, such as acetaminophen (APAP), are safe at recommended pharmacological doses but cause acute, dose-related DILI when overdosed. APAP hepatotoxicity is characterized by hepatocellular necrosis/apoptosis and liver failure that can be resolved or be fatal, but there are no reported clinical cases of APAP-induced liver fibrosis [8,11]
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