Positive regulation of hepatic miR-122 expression by HNF4α
Positive regulation of hepatic miR-122 expression by HNF4α
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DOI：
10.1016/j.jhep.2010.12.023
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发表时间：
2011-09-01
影响因子：
25.7
通讯作者：
Liang, Chih-Chuan
中科院分区：
文献类型：
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作者：
Li, Zhen-Ya;Xi, Yang;Liang, Chih-Chuan
背景和目标：miR-122是肝脏中最丰富的microRNA，调节代谢途径，包括胆固醇生物合成、脂肪酸合成和氧化。然而，关于调节肝脏中miR-122表达的机制知之甚少。方法：采用生物信息学分析、北方印迹、RT-PCR和5 '/3' RACE技术，分析miR-122的转录本结构、启动子区域和潜在的转录因子结合位点。采用报告基因分析结合miR-122启动子截短和位点突变的方法，在体外检测HNF 4 α对miR-122启动子的反式激活作用。进行ChIP和EMSA测定以确定HNF 4 α与miR-122启动子的结合。最后，通过强制表达和RNAi技术验证了HNF 4在体内外对miR-122表达的调控作用。结果：miR-122是由一个长剪接的初级转录物加工而成，该转录物由一个跨物种保守的远端上游启动子区域指导。我们解剖了这个启动子区域，并确定了肝脏富集的转录因子的假定结合位点，这些转录因子有助于调节miR-122的表达，包括肝细胞核因子4 α（HNF 4 α）的假定结合位点。我们证明HNF 4 α通过保守的DR-I元件与miR-122启动子区结合。我们观察到HNF 4 α对保守的miR-122启动子的DR-1元件依赖性激活作用，并且通过加入PGC 1 α可以进一步增强激活。使用过表达和敲低策略，我们发现HNF 4 α正调控miR-122在Huh 7细胞和小鼠liver.Conclusions表达：我们的研究结果表明，HNF 4 α是miR-122在肝脏中表达的关键调节因子。2011年欧洲肝脏研究协会。Elsevier B. V.出版，保留所有权利。
Background & Aims: 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 HNF4 alpha to miR-122-promoter in vitro. ChIP and EMSA assays were performed to determine HNF4 alpha 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 4 alpha (HNF4 alpha). We demonstrate that HNF4 alpha binds to the miR-122 promoter region through the conserved DR-I element. We observed the DR-1-element-dependent activation effect of HNF4 alpha on the conserved miR-122 promoter and the activation could be further enhanced by the addition of PGC1 alpha. Using overexpression and knockdown strategies, we show that HNF4 alpha positively regulates miR-122 expression in both Huh7 cells and the mouse liver.Conclusions: Our results suggest that HNF4 alpha is a key regulator of miR-122 expression in the liver. 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.