In silico and in vitro identification of microRNAs that regulate hepatic nuclear factor 4α expression

Anuradha Ramamoorthy, Lang Li, Andrea Gaedigk, L. DiAnne Bradford, Eric Benson, David A. Flockhart, Todd Skaar

Research output: Contribution to journalArticle

50 Citations (Scopus)

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 downregulated (∼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.

Original languageEnglish
Pages (from-to)726-733
Number of pages8
JournalDrug Metabolism and Disposition
Volume40
Issue number4
DOIs
StatePublished - Apr 2012

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MicroRNAs
Computer Simulation
Luciferases
Liver
3' Untranslated Regions
Cytochrome P-450 CYP2D6
Hep G2 Cells
Computational Biology
Transcription Factors
Down-Regulation
In Vitro Techniques
Gene Expression
Messenger RNA
Enzymes
Pharmaceutical Preparations
Genes
Proteins

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

Cite this

In silico and in vitro identification of microRNAs that regulate hepatic nuclear factor 4α expression. / Ramamoorthy, Anuradha; Li, Lang; Gaedigk, Andrea; Bradford, L. DiAnne; Benson, Eric; Flockhart, David A.; Skaar, Todd.

In: Drug Metabolism and Disposition, Vol. 40, No. 4, 04.2012, p. 726-733.

Research output: Contribution to journalArticle

Ramamoorthy, Anuradha ; Li, Lang ; Gaedigk, Andrea ; Bradford, L. DiAnne ; Benson, Eric ; Flockhart, David A. ; Skaar, Todd. / In silico and in vitro identification of microRNAs that regulate hepatic nuclear factor 4α expression. In: Drug Metabolism and Disposition. 2012 ; Vol. 40, No. 4. pp. 726-733.
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