Mapping the miRNA‐mRNA Interactome in Human Hepatocytes and Identification of Functional mirSNPs in Pharmacogenes

Abstract

MiRNAs regulate the expression of hepatic genes involved in pharmacokinetics and pharmacodynamics. Genetic variants affecting miRNA binding (mirSNPs) have been associated with altered drug response, but previously used methods to identify miRNA binding sites and functional mirSNPs in pharmacogenes are indirect and limited by low throughput. We utilized the high‐throughput chimeric‐eCLIP assay to directly map thousands of miRNA‐mRNA interactions and define the miRNA binding sites in primary hepatocytes. We then used the high‐throughput PASSPORT‐seq assay to functionally test 262 potential mirSNPs with coordinates overlapping the identified miRNA binding sites. Using chimeric‐eCLIP, we identified a network of 448 miRNAs that collectively target 11,263 unique genes in primary hepatocytes pooled from 100 donors. Our data provide an extensive map of miRNA binding of each gene, including pharmacogenes, expressed in primary hepatocytes. For example, we identified the hsa‐mir‐27b‐DPYD interaction at a previously validated binding site. A second example is our identification of 19 unique miRNAs that bind to CYP2B6 across 20 putative binding sites on the transcript. Using PASSPORT‐seq, we then identified 24 mirSNPs that functionally impacted reporter mRNA levels. To our knowledge, this is the most comprehensive identification of miRNA binding sites in pharmacogenes. Combining chimeric‐eCLIP with PASSPORT‐seq successfully identified functional mirSNPs in pharmacogenes that may affect transcript levels through altered miRNA binding. These results provide additional insights into potential mechanisms contributing to interindividual variability in drug response.

A case-control study of a combination of single nucleotide polymorphisms and clinical parameters to predict clinically relevant toxicity associated with fluoropyrimidine and platinum-based chemotherapy in gastric cancer

“…In addition, another regulator of gene expression are the Micro RNA (miRNAs), where genetic variations in miRNA binding sites are associated with an altered drug response [98]. In a similar vein, a recent publication by Powell et al reported the mapping of miRNA-mRNA interactions in several pharmacogenes, in fact, the authors identified an hsa-mir-27b-DPYD interaction at a previously validated binding site, which may suggest the existence of additional elements that contribute to the individualism of drug response [99].…”

Regulation of Drug Transport Proteins—From Mechanisms to Clinical Impact: A White Paper on Behalf of the International Transporter Consortium

“…Currently, no systematic data are available to identify the functional significance of miRNAs, and methods to identify miRNA binding sites are indirect and time consuming. To address this gap, application of the chimeric‐eCLIP assay recently described to map the hepatocyte miRNA‐mRNA interactome 117 should be explored to identify miRNAs binding to transporter mRNAs in hepatocytes and cells in other organs. Another class of regulatory RNAs that is understudied are the lncRNAs.…”

Analysis of the combined effect of rs699 and rs5051 on angiotensinogen expression and hypertension

“…We previously reported that (1) AGT mRNA is endogenously bound by miR‐122‐5p in human hepatocytes using the mir‐eCLIP assay and that (2) rs699 A > G significantly decreases reporter mRNA levels in hepatocytes using the high‐throughput functional screening assay PASSPORT‐seq 34 . Since microRNAs bind to and decrease mRNA abundance, our assay results suggest miR‐122‐5p decreases AGT mRNA more with the rs699 A > G variant.…”

“…The ViennaRNA suite version 2.4.14 38 was used to call RNAduplex with default settings to test the effect of rs699 A > G on binding strength. The miR‐122‐5p sequence was obtained online from miRbase (https://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000442), and the AGT region of rs699 was obtained from the PASSPORT‐seq oligo sequence used to clone the reporter construct 34 . This sequence included complementary primer ends which were included in the analysis to make sure miR‐122‐5p was not predicted to target these.…”

“…The mir‐eCLIP assay was performed in single‐donor primary hepatocytes obtained from Xenotech (lot HC2‐47) using the kit reagents and protocol provided by ECLIPSE Bioinnovations (catalog number not yet available). This protocol is developed based on the published single‐end seCLIP protocol 42 and is refined as previously described 34 . Pooled‐hepatocytes were used for Run 1 (previously completed), and data from this assay 34 was used.…”