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人肝脏特异性microRNA-122受转录因子FOXO1调控及其机制研究
【作者】 习杨；
【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2010， 博士
【摘要】 microRNA(miRNA)是一类长度约20～24 nt的非编码单链小分子RNA,它们通过与目标mRNA分子的3’非翻译区(3’-untranslated region,3’-UTR)互补匹配导致该mRNA分子的翻译受到抑制或影响其稳定性,从而调控下游的生物学过程。近年来,除了对microRNA产生的剪接机制和下游功能展开了深入而广泛的研究,对于microRNA初始转录本生成的转录调控研究也是方兴未艾。人miR-122是我们所关注的一个肝脏特异性小RNA。它的初始转录本来源于人18号染色体上hcr基因的转录,其成熟本在进化过程中高度保守。miR-122在肝脏发育过程中持续表达,且随肝脏细胞的分化成熟表达增高。研究表明：在病理状态下,miR-122促进丙型肝炎病毒(HCV)在肝细胞内复制,并与肝细胞肝癌(HCC)的发生、发展及转移等过程密切相关；在生理状态下,miR-122参与肝细胞表型,肝脏的分化,发育与代谢,以及肝细胞应急应答等基本生命活动过程。miR-122的沉默可导致小鼠血浆胆固醇和甘油三酯水平的显著降低,说明miR-122是肝脏脂肪代谢的重要调控者。FOXO1是哺乳动物叉头蛋白家族O亚族四个成员之一。叉头蛋白家族中,特别是FOXO1在整个机体的能量代谢中起着至关重要的作用。在饥饿状态下,肝脏通过FOXO1在促进糖异生酶类表达中的关键作用维持血糖的水平。而在进食后,胰岛素分泌增加,通过PI3K-Akt途径磷酸化FOXO1使其出核,部分抑制糖异生酶类在肝脏中的表达。除了直接调控代谢,FOXO1还在骨骼肌和脂肪组织这两大维系能量稳态平衡的主要器官的形成过程中起重要作用。另外,FOXO1的表达可促进抗氧化应激的基因表达,从而起到细胞保护功能。FOXO1也能促进碳水化合物向脂肪酸的转换,使其在饥饿状态下成为主要的能量来源。本研究旨在探索糖脂代谢重要因子FOXO1对miR-122的转录调控作用及其调控机制。首先,我们利用UCSC网站和ECR browser等在线分析软件对miR-122成熟本上游5kb的片段进行生物信息学的分析,发现在之前本室鉴定的miR-122核心启动子区存在一个FOXO1的保守结合位点。然后利用瞬时转染和筛选稳定克隆两种办法将FOXO1的野生型表达质粒FOXO1-WT和有着三个磷酸化位点突变的核内组成型表达质粒FOXO1-CA及空载体对照在肝细胞系Huh7中表达,发现野生型FOXO1蛋白对miR-122的转录呈现正调控模式,而核内组成型表达的FOXO1蛋白对miR-122的转录呈现负调控模式。接着,运用双荧光素酶报告基因系统确定了FOXO1对miR-122上游启动子区存在转录调控作用,并利用miR-122上游调控区的片段截短和突变实验来确定了FOXO1调控miR-122转录的具体位点是miR-122核心启动子区的IRE(胰岛素应答元件)元件。最后,我们利用鉴定转录因子的经典方法凝胶迁移速率实验(EMSA)和染色质免疫共沉淀实验(ChIP)分别从体外和体内进一步确定了FOXO1可以直接结合于miR-122核心启动子区的IRE位点。同时发现FOXO1的这种结合在血清剥夺后的低营养状态下增强而在回复血清后的高营养状态下减弱,并且它与HNF4对该IRE位点的结合是竞争性的。综上所述,我们的研究首次发现FOXO1可作为miR-122的转录因子,通过结合到miR-122核心启动子区的IRE位点来调控miR-122初始转录本的转录。这种结合受营养状态密切相关的信号通路的影响,同时也受到HNF4对该位点结合的竞争,可最终参与到肝脏脂代谢的精细调控过程中。
【Abstract】 MicroRNA(miRNA) is a kind of 20-24nt non-coding single strand small RNAs. They have been shown to extensively involve in the translational inhibition and/or degradation of a wide variety of mRNA molecules through imperfect pairing to their 3’-UTR (3’-untranslated region). Besides the biogenesis and functional research of microRNAs themselves, transcriptional control of pri-microRNA has attracted increasing attention.The evolutionary highly conserved miR-122 becomes our focus as the dominant liver miRNA with comprehensive roles in the pathophysiology of the organ. MiR-122 was found promotes HCV replication in hepatocyte, and tightly related to the biogenesis, development and the migration of Hepatocellular carcinoma (HCC). Physiologically, miR-122 participates in the hepatocyte phenotype, the differentiation, development and metabolism of the liver, and the hepatocyte response to stimuli. Role of miR-122 in lipid metabolism was emphasized by the in vivo silencing of miR-122 with stably modified anti-sense RNA, which led to significant decrease of plasma cholesterol and triglyceride in mice.We have previously characterized the pri-hsa-miR-122 transcription from her gene at 18q21.31 of human genome and also identified a conserved HNF4 binding site within the pri-hsa-miR-122 promoter that is responsible for the liver specificity of miR-122 expression. Further exploration of the pri-hsa-miR-122, especially its promoter region will better fit the miRNA into a regulatory network comparable to its versatile pathophysiological functions.FOXO1 represents one of the four members in the mammalian forkhead box class O (FoxO) family. Forkhead proteins, FoxOl in particular, play significant roles in energy homeostasis of the body. In the fasted state, the liver is primarily responsible for maintaining glucose levels, with FoxOl playing a key role in promoting the expression of gluconeogenic enzymes. Following feeding, pancreatic beta cells secrete insulin, and partially suppress gluconeogenic enzyme expression in the liver by activating PI3K-AKT mediated FoxO1 exportation to the cytosol. It has also been found that FoxO1 regulated lipid/energy metabolism by promoting the switch from carbohydrate to fatty acid as the major energy source during starvation. In addition to directly regulating of metabolism, FoxO1 also plays a role in the formation of both adipose tissue and skeletal muscle, two major organs that are critical for maintaining energy homeostasis.In this study, we explored the possibility that FOXO1, the key transcriptional factor in glucose-lipid metabolism, regulates the transcription of miR-122, which also significantly involved in lipid metabolism. After scrutiny of the first 5kb upstream to the mature miR-122 with bioinformatics tools including UCSC and ECR browser, a conserved FoxO1 binding site was located in the miR-122 core promoter identified previously by our lab. Plasmids carrying either FOXO1-WT or FOXO1-CA(a constant active mutant of FoxO1) and the vector pEGFP-N2 itself were then transfected separately into Huh7cell line, both transiently and stably. The result indicated differentiated regulatory effects of FOXO1-WT, which positively regulated the transcription of miR-122, and FOXO1-CA, which regulated it negatively. The deviation in the interpretation of FoxO1 effects on miR122 implys potential dose-dependent regulatory pattern that demands further experimental evidence. Dual Luciferase Reporter Gene Assay for truncation and mutation of miR-122 promoter identified that the precise binding site of FOXO1 as the insulin response element (IRE) within miR-122 core promoter. The subsequent EMSA and ChIP assays demonstrated the binding of FOXO1 to the IRE site both in vitro and in vivo. The observed FoxO1 binding increased in low nutrient status imposed by serum deprivation and decreased upon serum recovery. Besides, the binding of FOXO1and HNF4 were found reciprocal with their binding sites adjacent to each other at the IRE site.Altogether, our study suggests FOXO1 as a new regulator to the transcription of pri-miR-122 through the IRE site of its core promoter. FoxO1 together with the previously identified HNF4, synergized and antagonized, in providing a delicate regulatory mechanism through which miR-122 may response to the nutrient status and other ambient stimuli of the host cells.