title: Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays.
abstract: OBJECTIVE
  To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions.
  
  
  METHODS
  HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. Total RNA and mRNA of quiescent HSCs, and culture-activated HSCs were extracted, quantified and reversely transcripted into cDNA. The global gene expression profile was analyzed by microarray with Affymetrix rat genechip. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Microarray data were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi. The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl(4))-induced fibrosis rat model was also analyzed with Western blotting.
  
  
  RESULTS
  Of the 28 700 genes represented on this chip, 2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate. Of these, 1396 genes were upregulated, while 1170 genes were downregulated in culture-activated HSCs. These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms. The most enriched GO terms included response to wounding, wound healing, regulation of cell growth, vasculature development and actin cytoskeleton organization. KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs. Wnt5a was significantly increased in culture-activated HSCs as compared with quiescent HSCs. qRT-PCR validated the microarray data. Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation, downregulated expressions of type I collagen and transforming growth factor-β1. Wnt5a was upregulated in the fibrotic liver of a CCl(4)-induced fibrosis rat model.
  
  
  CONCLUSIONS
  Wnt5a is involved in the activation of HSCs, and it may serve as a novel therapeutic target in the treatment of liver fibrosis.
authors.affiliation: Department of Gastroenterology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200120, China. xiongwujun@hotmail.com
authors.forename: Wu-Jun
authors.initials: WJ
authors.lastname: Xiong
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Hu
authors.forename: Yi-Cheng
authors.initials: YC
authors.lastname: Jian
authors.forename: Li-Jing
authors.initials: LJ
authors.lastname: Wang
authors.forename: Ming
authors.initials: M
authors.lastname: Jiang
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Yi
authors.initials: Y
authors.lastname: He
mesh_terms: D000818:Animals; D020013:Calcium Signaling; D002251:Carbon Tetrachloride / Q000633:toxicity; D019295:Computational Biology; D020869:Gene Expression Profiling; D055166:Hepatic Stellate Cells / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D051153:Wnt Proteins / Q000502:physiology*; D060449:Wnt Signaling Pathway; D000071818:Wnt-5a Protein
chemical_list: D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C491837:Wnt5a protein, rat; D002251:Carbon Tetrachloride