title: Overexpression of miR-122 promotes the hepatic differentiation and maturation of mouse ESCs through a miR-122/FoxA1/HNF4a-positive feedback loop.
abstract: OBJECTIVE
  microRNA-122 is the only identified liver-specific miRNA and plays a crucial role in liver development, maintenance of hepatic homeostasis as well as tumourigenesis. In our previous differentiation of ESCs into hepatocytes, microRNA-122 (miR-122) was expressed at a relatively low level. Here, we aim to elucidate the effect and underlying mechanisms of miR-122 during differentiation of ESCs into hepatocytes.
  
  
  METHODS
  Mouse ESCs were initially induced towards HPCs by activin A, FGF-4 and sodium butyrate and were subsequently transfected with a recombinant adenovirus expressing vector pAV.Ex1d-CMV>miR-122/IRES/eGFP 9 days after induction. Cells were analysed by real-time PCR, immunofluorescence, flow cytometry, microscopy and functional assays. Furthermore, microarray analysis was performed.
  
  
  RESULTS
  We demonstrated that overexpression of miR-122 could effectively promote hepatic differentiation and maturation, as assessed by morphological and functional tests. The microarray analysis revealed that 323 genes were down-regulated, whereas 59 were up-regulated. Particularly, two liver-specific transcription factors, FoxA1 and HNF4a, were significantly up-regulated. Moreover, the expression of E-cadherin was dramatically increased and the proliferation of HPCs was suppressed, whereas knockdown of FoxA1 reduced E-cadherin expression and increased the proliferation of HPCs. In addition, the expression levels of FoxA1, HNF4a and E-cadherin in time-course transfection experiments with miR-122 were not significantly increased except in cells in which transfection with miR-122 occurred 9 days after induction.
  
  
  CONCLUSIONS
  Overexpression of miR-122 at an appropriate stage could promote hepatic differentiation and maturation by regulating the balance between proliferation and differentiation, as well as the balance between EMT and MET, partially through a miR-122/FoxA1/HNF4a-positive feedback loop.
authors.affiliation: Department of Pediatric Surgery, The Memorial Hospital of Sun Yat-Sen University, Guangzhou, China.
authors.forename: Xiao-Geng
authors.initials: XG
authors.lastname: Deng
authors.forename: Rong-Lin
authors.initials: RL
authors.lastname: Qiu
authors.forename: Yao-Hao
authors.initials: YH
authors.lastname: Wu
authors.forename: Zhi-Xi
authors.initials: ZX
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Xie
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Jia-Jia
authors.initials: JJ
authors.lastname: Zhou
authors.forename: Le-Xiang
authors.initials: LX
authors.lastname: Zeng
authors.forename: Jing
authors.initials: J
authors.lastname: Tang
authors.forename: Anu
authors.initials: A
authors.lastname: Maharjan
authors.forename: Jie-Min
authors.initials: JM
authors.lastname: Deng
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D025461:Feedback, Physiological / Q000502:physiology*; D005434:Flow Cytometry; D051541:Hepatocyte Nuclear Factor 3-alpha / Q000378:metabolism; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D046228:Microarray Analysis; D008856:Microscopy, Fluorescence; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C496500:Foxa1 protein, mouse; D051541:Hepatocyte Nuclear Factor 3-alpha; D051557:Hepatocyte Nuclear Factor 4; C496593:Hnf4a protein, mouse; D035683:MicroRNAs; C544354:Mirn122 microRNA, mouse
keywords: differentiation; embryonic stem cells; hepatocytes; mesenchymal-to-epithelial transition; miR-122