In silico and in vitro identification of microRNAs that regulate hepatic nuclear factor 4α expression.
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.
Department of Medicine, Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Anuradha
A
Ramamoorthy
Lang
L
Li
Andrea
A
Gaedigk
L DiAnne
LD
Bradford
Eric A
EA
Benson
David A
DA
Flockhart
Todd C
TC
Skaar
D020413:3' Untranslated Regions / Q000235:genetics; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005838:Genotype; D006367:HeLa Cells; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics* / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics / Q000502:physiology*; D004364:Pharmaceutical Preparations / Q000378:metabolism; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
D020413:3' Untranslated Regions; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; C522425:MIRN34 microRNA, human; C544530:MIRN449 microRNA, human; D035683:MicroRNAs; D004364:Pharmaceutical Preparations; D012333:RNA, Messenger; D008156:Luciferases; D019389:Cytochrome P-450 CYP2D6