Positive regulation of hepatic miR-122 expression by HNF4α.
OBJECTIVE
miR-122 is the most abundant microRNA in the liver and regulates metabolic pathways including cholesterol biosynthesis, fatty acid synthesis, and oxidation. However, little is known about mechanisms that regulate the expression of miR-122 in the liver. The aim of this study was to identify key transcriptional regulators for miR-122 expression through intensively studying its primary transcript and promoter region.


METHODS
Bioinformatics analysis, Northern blotting, RT-PCR, and 5'/3' RACE were performed to analyze miR-122 primary transcript structure, its promoter region, and potential transacting factor binding sites. Reporter gene assays integrated with truncation and site-mutation in miR-122 promoter were performed to determine the trans-activation effect of HNF4α to miR-122-promoter in vitro. ChIP and EMSA assays were performed to determine HNF4α binding to miR-122 promoter. Finally, forced expression and RNAi were performed to verify the regulatory roles of HNF4 to miR-122 expression in vitro and in vivo.


RESULTS
Here, we show that miR-122 is processed from a long spliced primary transcript directed by a distal upstream promoter region conserved across species. We dissected this promoter region and identified putative binding sites for liver-enriched transcriptional factors that contribute to the regulation of miR-122 expression, including a putative binding site for hepatocyte nuclear factor 4α (HNF4α). We demonstrate that HNF4α binds to the miR-122 promoter region through the conserved DR-I element. We observed the DR-1-element-dependent activation effect of HNF4α on the conserved miR-122 promoter and the activation could be further enhanced by the addition of PGC1α. Using overexpression and knockdown strategies, we show that HNF4α positively regulates miR122 expression in both Huh7 cells and the mouse liver.


CONCLUSIONS
Our results suggest that HNF4α is a key regulator of miR-122 expression in the liver.
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Zhen-Ya
ZY
Li
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Yang
Y
Xi
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Wen-Nan
WN
Zhu
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Chao
C
Zeng
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Zhu-Qin
ZQ
Zhang
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Zhi-Chen
ZC
Guo
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
De-Long
DL
Hao
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Guang
G
Liu
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Lei
L
Feng
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Hou-Zao
HZ
Chen
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Feng
F
Chen
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Xiang
X
Lv
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China. Electronic address: liudp@pumc.edu.cn.
De-Pei
DP
Liu
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Chih-Chuan
CC
Liang
D000818:Animals; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006367:HeLa Cells; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014158:Transcription, Genetic
C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D014157:Transcription Factors