Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Apr 21, 2012; 18(15): 1745-1752
Published online Apr 21, 2012. doi: 10.3748/wjg.v18.i15.1745
Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays
Wu-Jun Xiong, Li-Juan Hu, Yi-Cheng Jian, Li-Jing Wang, Ming Jiang, Wei Li, Yi He
Wu-Jun Xiong, Li-Juan Hu, Yi-Cheng Jian, Li-Jing Wang, Ming Jiang, Wei Li, Yi He, Department of Gastroenterology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200120, China
Author contributions: Xiong WJ designed the research; Hu LJ, Jian YC, Wang LJ, Jiang M, Li W, He Y performed the research; Hu LJ wrote the paper.
Supported by Research Grant for Health Science and Technology of Pudong Health Bureau of Shanghai, No. PKJ2009-Y16
Correspondence to: Wu-Jun Xiong, Associated Professor, Department of Gastroenterology, Shanghai East Hospital Affiliated to Tongji University, 150 Jimo Road, Shanghai 200120, China. xiongwujun@hotmail.com
Telephone: +86-21-38804518-2063 Fax: +86-21-58798999
Received: June 9, 2011
Revised: September 10, 2011
Accepted: February 27, 2012
Published online: April 21, 2012
Abstract

AIM: 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 (CCl4)-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 CCl4-induced fibrosis rat model.

CONCLUSION: Wnt5a is involved in the activation of HSCs, and it may serve as a novel therapeutic target in the treatment of liver fibrosis.

Keywords: Hepatic stellate cells, Wnt5a, Microarray, Bioinformatics analyses, Liver fibrosis