Man Liver-specific Micrornas-122 By The Transcription Factor Foxo1 Regulation And Its Mechanism

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.