Serum‐derived Extracellular Vesicles as Diagnostic Biomarkers for Non‐alcoholic Fatty Liver Disease

Abstract

IntroductionNon‐alcoholic fatty liver (NAFL) disease is a highly prevalent chronic liver disease with variable severity, from simple steatosis to steatohepatitis (NASH). NASH involves hepatocyte death, inflammatory infiltration and fibrosis, and may progress to cirrhosis or hepatocellular carcinoma. Liver biopsy is currently the only way to definitively diagnose NASH, but this simply provides a static image and due to the highly invasive nature of the procedure, poses risks to patients and is not practical for longitudinal evaluation. Circulating populations of small extracellular vesicles (sEVs), including exosomes (30–150nm) and microvesicles (50–1000nm), are known to exhibit alterations in concentration and molecular cargo in disease states, including NAFL and NASH. Thus, they present the lucrative opportunity to not only diagnose and stage disease in a minimally invasive manner, but also track the efficacy of therapeutic interventions.AimThis study aimed to evaluate the performance of miRNA, derived from global circulating sEVs or hepatocyte‐specific EVs, in distinguishing patients with NAFL and NASH from healthy individuals.MethodsSerum was obtained from healthy volunteers and patients with NAFL and NASH and ExoQuickTM precipitation solution was used to isolate global sEV samples. Magnetic DynaBeads conjugated to anti‐asialoglycoprotein receptor 1 antibody were used for immunoprecipitation of hepatocyte‐specific EVs from global samples. Total RNA was isolated from EV samples using TRIzol reagent and reverse transcribed with TaqMan miRNA RT kit. Expression of miR ‐122, ‐192, ‐128‐3p, ‐451 and ‐16 was determined by RT‐qPCR using TaqMan small RNA assays. Predictive performance of miRNA biomarkers was evaluated by receiver operating characteristic (ROC) analysis.ResultsExpression of liver miRNA (miR ‐122, ‐192, ‐128‐3p) given as ratios to non‐specific EV miRNA (‐451, ‐16) in global circulating EVs was generally greater in patients with NAFL and NASH. In distinguishing all disease from healthy, excellent performance was exhibited by miR‐128‐3p/‐451 and miR‐192/‐451 (AUROC = 0.925 and 0.882, respectively). miR‐122 ratio expression was relatively poor in global EVs but markedly improved in liver‐specific EVs, up to AUROC = 1.000.ConclusionWe provide a proof of principle for the use of EV‐derived miRNA ratio biomarkers in minimally invasive NAFLD diagnosis and describe a method for extracting liver‐specific EVs for this application.Support or Funding InformationNHMRC project grant (1158210) Pfizer Investigator Initiated project grantReceiver operating characteristic (ROC) curves for miR‐122 ratios derived from liver‐specific extracellular vesicles (EVs) in predicting all NAFLD and only fatty liver.Figure 1