Expression Analysis of Exosomal MicroRNAs in Chlorpromazine Treated Human Hepatic Spheroids: A Promising Tool for Novel Drug-Induced Liver Injury Biomarker Discovery

Abstract

Introduction: Risk management and mitigation of new drug candidates that induce drug-induced liver injury (DILI) in the clinic is a pivotal task to protect patients and save money during the drug development process. Nevertheless, clinical DILI risk assessment with conventionally available liver biomarkers comes with high failure rates. MicroRNAs (miRNAs) are short noncoding RNA molecules known to regulate mRNA expression. Actively secreted miRNAs which are circulating in patient blood can provide important pathway information involving specific DILI-inducing mechanisms and, therefore, have potential as early diagnostic biomarkers. Materials and Methods: In this study, the exosomal miRNA response of human primary hepatic spheroids treated with toxic concentrations of Chlorpromazine for 3 or 8 days was analyzed. The miRNA response of the hepatic spheroids was analyzed by quantifying a total of 752 miRNAs in the culture medium by quantitative polymerase chain reaction. Results: Clustering and pathway enrichment analysis of the miRNA response revealed several differentially expressed miRNAs involved in the regulation of signaling pathways that are toxicologically relevant, including apoptosis. This exosomal miRNA response was validated by a caspase 3/7 activity assay which confirmed an induction of apoptosis in hepatic spheroids treated with Chlorpromazine. Conclusion: Our results suggest that human hepatic spheroids are a promising test system for the discovery of secreted miRNA signatures. This could become a novel tool for discovery of human in vivo relevant compound-specific DILI biomarkers, inducing understanding mechanisms. In addition, miRNA biomarkers which can be monitored directly in the patient blood can become valuable tools to assist clinicians in risk management.