In this study, we performed large-scale integration of genomic information, transcriptome data, and miRNAome profiling in human liver. The resulting integrative map offers the possibility of identifying key regulatory pathways involved in disease biology and therapeutic outcome. Genome annotation of global mRNA and miRNA expression in this tissue should facilitate explorations of the complex interrelationships between genetic variation, the regulome consisting of the regulatory nodes and networks underlying biological function, and complex traits (pathophysiology and pharmacogenomic traits). The resource we have created expands on earlier studies of the heritability of miRNA expression in (transformed) lymphocytes  and of the utility of eQTL mapping in a variety of tissues for the identification of disease-associated genes [33, 34].
This study presents a genome-wide analysis of miRNA expression in human liver, with a primary focus on understanding miRNA regulatory effects on the transcriptome, but also a special emphasis on obtaining pharmacogenomic insights from an exploration of gene regulation in the tissue of key importance for drug metabolism and excretion. Consistent with studies that show that miRNAs target a third of the genome , we found that 275 miRNAs are inversely correlated (FDR < 0.05) with the expression levels of 5,767 genes. This finding implies that mRNA expression in liver is likely to be altered by multiple miRNAs. In particular, we identified several miRNAs that significantly downregulate (FDR < 0.05) the so-called VIP genes, which are key pharmacogenes involved in modulating response to one or more drugs; these miRNAs are thus likely to have a significant pharmacodynamic or pharmacokinetic influence on drug response phenotypes. For example, miR-23b, which has been implicated in liver stem cell differentiation , significantly downregulates the expression of multiple VIP genes (FDR < 0.05), including F5, ADRB1, GSTP1, KCNH2, KCNJ11, NQO1, and PTGIS. In addition, we have shown that these important pharmacogenes are significantly enriched for large miRNA regulatory effects in liver, compared to genome-wide expectation.
We found, in a gene set enrichment analysis, that the genes with the most significant negative correlations with miRNAs in liver were enriched for cell adhesion molecules. Regulation of cell adhesion molecules has been shown to play a role in the pathogenesis of many human diseases  as well as in normal cellular and organismal homeostasis . A growing body of literature (for review, see Commentary in ) has demonstrated the crucial role of miRNAs in four major adhesion processes: cytoskeletal dynamics, cell-cell adhesion, cell-matrix adhesion, and extracellular matrix; interestingly, as Robert Weinberg et al. noted, those miRNAs that target genes belonging to more than one of these adhesion processes are notably the same miRNAs that have been implicated in various human diseases [38–40].
Our study demonstrates that miRNA expression in human liver has a significant genetic component. Most investigations of the effect of genetic variation on miRNAs have so far been focused on genetic influence (e.g., 3′ UTR SNPs) on miRNA target site recognition. Our study sought evidence for the role of genetic variation in modulating miRNA expression variation.
Importantly, our findings can be integrated into studies of complex phenotypes from genome-wide association studies and sequencing efforts. We investigated published associations between genetic variation and disease susceptibility or quantitative traits from the NHGRI repository. For example, we identified two SNPs, rs4598195 and rs4730276, that have been found to be associated with ulcerative colitis [41, 42] and that predict the expression of hsa-miR-629*; remarkably, hsa-miR-629 has been independently found to be differentially expressed in ulcerative colitis . Two independent SNPs, rs7191888 on chromosome 16 and rs10259085 on chromosome 7, are reported associations with multiple sclerosis  and associated with the expression of hsa-miR-126* and hsa-miR-126 respectively in our dataset. A SNP rs6085920 has been associated with serum uric acid  and is associated with the expression of miRNA hsa-miR-141, which has been proposed as a therapeutic target for the prevention of progressive kidney disease .
In a similar vein, we systematically investigated published clinical pharmacogenetic associations for their role in regulating miRNA expression. A SNP rs4888024 was found to be associated with end-of-induction minimal residual disease in childhood acute lymphoblastic leukemia from 2 independent cohorts and higher methotrexate clearance . Leukemias with rearrangement of the MLL gene have been shown to be characterized by the absence of hsa-miR-340 expression . Our miR-eQTL data support a relationship (p = 8.5x10-4) between rs4888024 and the expression of hsa-miR-340. Furthermore, a SNP rs730012 (in LTC4S) has been found to be associated with exacerbation rates in asthma patients treated with montelukast . Our data support a relationship between this SNP and the expression of miR-146b (p = 3.89x10-4), which has been implicated in asthma pathogenesis in murine models of acute and chronic asthma . As the detailed mechanisms underlying numerous genotype-phenotype correlations in both disease genomics and pharmacogenomics remain largely unknown, our study provides important hypotheses for future investigations. To this end, we make the results of our study available to the scientific community through an online public resource .
Using Q-PCR, we were able to confirm the expression of two miRNAs and two mRNAs, the inverse correlations between these miRNAs and mRNAs, and an identified miR-eQTL, despite the limited sample size due to the availability of RNA. Although the biological interactions between the miRNAs and the mRNAs will require extensive experimental validation (e.g. cloning, transfection etc.), our study generates, in aggregate, numerous hypotheses that warrant continued investigations and that may have substantial impact on the study of human diseases as well as on pharmacogenetics.