Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Feb 8, 2017; 9(4): 191-208
Published online Feb 8, 2017. doi: 10.4254/wjh.v9.i4.191
Regulation of hepatic microRNA expression by hepatocyte nuclear factor 4 alpha
Hong Lu, Xiaohong Lei, Jerry Liu, Curtis Klaassen
Hong Lu, Xiaohong Lei, Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, United States
Jerry Liu, Curtis Klaassen, Department of Medicine, University of Kansas Medical Center, Kansas City, KS 66160, United States
Author contributions: Lu H wrote the paper; Lu H, Lei X and Liu J performed the experiments and analyzed the data; Lu H and Klaassen C conceived and designed the experiments.
Supported by NIH grant ES019487 in part.
Institutional animal care and use committee statement: All animal procedures in the study were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center. The animal protocol was designed to minimize the pain or distress to the mice. Agematched young-adult HNF4α Liv-KO mice and their wild-type control littermates were fed rodent chow (#8064, Teklad; Harlan, Indianapolis, IN). Mice were housed at an ambient temperature of 22 °C with alternating 12-h light/dark cycles and allowed water and feed ad libitum.
Conflict-of-interest statement: The authors have no conflict of interest to declare.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Hong Lu, PhD, Assistant Professor, Department of Pharmacology, SUNY Upstate Medical University, 750 E Adams ST, Syracuse, NY 13210, United States. luh@upstate.edu
Telephone: +1-315-4647978 Fax: +1-315-4648008
Received: July 30, 2016
Peer-review started: August 2, 2016
First decision: September 8, 2016
Revised: October 2, 2016
Accepted: December 1, 2016
Article in press: December 2, 2016
Published online: February 8, 2017
Processing time: 191 Days and 3.2 Hours
Abstract
AIM

To uncover the role of hepatocyte nuclear factor 4 alpha (HNF4α) in regulating hepatic expression of microRNAs.

METHODS

Microarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4α expression in liver (Hnf4α-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4α, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4α on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3’UTR) of two genes in human hepatoma cells.

RESULTS

Microarray data indicated that most microRNAs remained unaltered by Hnf4α deficiency in Hnf4α-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4α-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4α-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4α directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4α markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3’UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1.

CONCLUSION

HNF4α is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4α may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.

Keywords: Liver; Hepatocyte nuclear factor 4 alpha; Knockout; Mice; Human; miR-122; miR-192; miR-194; miR-101; miR-802

Core tip: Hepatocyte nuclear factor 4 alpha (HNF4α) is a liver-enriched master regulator of liver development and function. HNF4α plays a key role in regulating hepatic transcriptome and epigenome. However, little was known about the role of HNF4α in regulating hepatic expression of microRNAs, essential modulators of the transcriptome and epigenome. Results from this study uncover species differences and similarities between humans and mice in the role of HNF4α in regulating hepatic expression of certain important microRNAs. Such novel knowledge will help understand the role of HNF4α in post-transcriptional regulation of gene expression and maintenance of the normal epigenome and physiology in mouse and human liver.