Identification of miR-199a-5p, miR-214-3p and miR-99b-5p as Fibrosis-Specific Extracellular Biomarkers and Promoters of HSC Activation

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.

Therapeutic delivery of microRNA-125a-5p oligonucleotides improves recovery from myocardial ischemia/reperfusion injury in mice and swine

“…However, considering that intramyocardial injection is not convenient in clinical treatment, how to improve the targeted efficacy and operational availability of microRNAs delivery in MI or the I/R myocardium requires further investigation. Meanwhile, some reports are incompatible with our findings regarding the functions of miR-125a-5p 55 - 58 . These inconsistencies may be caused by different disease models, animal species, and experimental conditions.…”

Systematic review of overlapping microRNA patterns in COVID-19 and idiopathic pulmonary fibrosis

“…Lino Cardenas et al have shown that miR-199a-5p pulmonary expression was significantly increased in IPF patients, and demonstrated that this miRNA behaves as a major mediator of lung fibrosis by promoting the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts [ 61 ]. It has been shown that miR-199a-5p is also increased during liver fibrosis and that miR-199a-5p plays a role in hepatic stellate cell activation, promoting α-SMA production and fibrosis progression [ 90 ].…”

Methotrexate-Induced Liver Injury Is Associated with Oxidative Stress, Impaired Mitochondrial Respiration, and Endoplasmic Reticulum Stress In Vitro

“…In 2D HepaRG, 100–150,000 nM MTX induced the accumulation of neutral lipids and reduced the viability to 70% [ 43 ]. Previous results from our laboratory demonstrated that ten days of exposure to 30,000 nM MTX induced fibrosis in a 3D in vitro model using HepaRG, hTERT-HSC, and THP-1, thus we set 30,000 nM MTX as the upper treatment limit [ 44 ]. For the lower treatment limit, we used 7 nM, a subtoxic concentration.…”