Abstract
Hepatic nuclear factor 4α (HNF4A) is a nuclear transcription factor that regulates the expression of many genes involved in drug disposition. To identify additional molecular mechanisms that regulate HNF4A, we identified microRNAs (miRNAs) that target HNF4A expression. In silico analyses suggested that HNF4A is targeted by many miRNAs. We conducted in vitro studies to validate several of these predictions. With use of an HNF4A 3′-untranslated region (UTR) luciferase reporter assay, five of six miRNAs tested significantly down-regulated (∼20–40%) the luciferase activity. In HepG2 cells, miR-34a and miR-449a also down-regulated the expression of both the HNF4A protein and an HNF4A target gene, PXR (∼30–40%). This regulation appeared without reduction in HNF4A mRNA expression, suggesting that they must be blocking HNF4A translation. Using additional bioinformatic algorithms, we identified polymorphisms that are predicted to alter the miRNA targeting of HNF4A. Luciferase assays indicated that miR-34a and miR-449a were less effective in regulating a variant (rs11574744) than the wild-type HNF4A 3′-UTR. In vivo, subjects with the variant HNF4A had lower CYP2D6 enzyme activity, although this result was not statistically significant (p = 0.16). In conclusion, our findings demonstrate strong evidence for a role of miRNAs in the regulation of HNF4A.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant 1R01-GM088076] (to T.C.S., A.G., L.L.); the National Institutes of Health Pharmacogenomics Research Network [Grant 5U01-GM061373-07] (to D.A.F.); the National Institutes of Health Agency for Healthcare Research and Quality [Grant R01-HS019818CM]; the Indiana University Cancer Center; and a Department of Defense predoctoral fellowship [Grant BC083078] (to A.R.).
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ABBREVIATIONS:
- HNF4A
- hepatic nuclear factor 4α
- miRNA
- microRNA
- 3′-UTR
- 3′-untranslated region
- SNP
- single nucleotide polymorphism
- miRSNP
- microRNA SNP
- bp
- base pair
- PCR
- polymerase chain reaction
- snRNA
- small nuclear RNA
- cel-miR
- Caenorhabditis elegans microRNA
- hsa-miR
- Homo sapiens microRNA
- RT
- reverse transcriptase
- siRNA
- small interfering RNA
- PXR
- pregnane X receptor
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- DM/DX
- dextromethorphan/dextrorphan.
- Received June 23, 2011.
- Accepted January 9, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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