In Vitro Validation of miRNA-Mediated Gene Expression Linked to Drug Metabolism

Abstract

Pharmacogenomic approaches used to investigate how genes affect drug responses are critical for designing personalized therapies aimed at maximizing efficacy and minimizing adverse effects. Drug efficacy is often dependent on the sequence and expression levels of drug target genes or those involved in the metabolism and transport of the therapeutic agent. Expression of these genes, in turn, is negatively regulated by small noncoding miRNAs. The levels of miRNAs in bodily fluids have been studied extensively as potential diagnostic and prognostic biomarkers. Studies have shown that miRNAs regulate multiple genes and sequence homology is used to predict which genes are subject to regulation by a particular miRNA. Once a gene is identified as a potential target for an miRNA of interest, experiments are undertaken to confirm that the miRNA interacts with the target gene and can alter its level of expression and/or its activity. For example, the differential expression of miRNAs in whole blood obtained from good and poor responders to ketamine has been reported both prior to, and following treatment for complex regional pain syndrome. In this case, hsa-miR-548d-5p was significantly lower in poor responders relative to good responders. This miRNA was predicted to target UDP-glucuronyl transferase 1A1 (UGT1A1), a key drug metabolizing enzyme. Described in this unit are protocols used to confirm miR-548d-5p-mediated UGT1A1 regulation. The approaches described can be employed broadly for the validation of miRNA-mediated negative regulation of any gene. Determining miRNA-mediated regulation of enzymes and transporters affecting drug metabolism is a critical step in designing personalized therapy and for understanding the mechanisms responsible for variations in the responses to therapeutic agents. © 2017 by John Wiley & Sons, Inc.