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. 2012 Apr 21;18(15):1745-52.
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  1 , Li-Juan Hu, Yi-Cheng Jian, Li-Jing Wang, Ming Jiang, Wei Li, Yi He
Affiliations

Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays

Wu-Jun Xiong et al. World J Gastroenterol. .

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 (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.

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: Bioinformatics analyses; Hepatic stellate cells; Liver fibrosis; Microarray; Wnt5a.

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Figures

Figure 1
Figure 1
Validation of microarray data by quantitative real-time polymerase chain reaction. Comparison between expression values of five selected differently expressed genes estimated by quantitative real-time polymerase chain reaction (qRT-PCR) and gene expression data. The detected changes measured by qRT-PCR reflected different changes in gene expression between quiescent and activated hepatic stellate cells obtained by microarray analysis. SD: Sprague Dawley; CTGF: Connective tissue growth factor; CamKII: Calcium calmodulin mediated kinaseII.
Figure 2
Figure 2
Wnt signaling pathway participated in the activation of hepatic stellate cells. 1Differentially expressed genes revealed by microarray. TGF-β1: Transforming growth factor-β1; PKC: Protein kinase; CamKII: Calcium calmodulin mediated kinase II; LRP: Low density lipoprotein receptor-related protein; GBP: Paternally expressed 12; APC: Adenomatous polyposis coli; CBP: CREB binding protein; PKC: Protein kinase C; NFAT: Nuclear factor of activated T cells; PLC: Phospholipase c.
Figure 3
Figure 3
Expression levels of Wnt5a proteins in three groups detected by Western blotting. 1: Control; 2: Negative siRNA; 3: Wnt5a ShRNA.
Figure 4
Figure 4
Knock down of Wnt5a decreases the cell proliferation. The role of Wnt5a in regulating LX-2 proliferation was determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Compared with the negative siRNA and control group, Wnt5a ShRNA could inhibit the proliferation of LX-2, aP < 0.05.
Figure 5
Figure 5
Knockdown of Wnt5a decreases expressions of α-1 type I collagen and transforming growth factor-β1. Experiments were repeated three times and representative results are shown. Compared with the negative siRNA, vector and control groups, the expressions of α-1 type I collagen (COL1A1) and transforming growth factor-β1 (TGF-β1) mRNA were downregulated remarkably in the Wnt5a shRNA group (P < 0.05). 1: Wnt5a ShRNA multiplicity of infection (MOI) 20; 2: Wnt5a ShRNA MOI 10; 3: Negative siRNA; 4:Vector; 5: Control.
Figure 6
Figure 6
Expression of Wnt5a in fibrotic livers determined with Western blotting. The bands were semi-quantitatively evaluated by densitometric analysis. Wnt5a protein was upregulated as significantly as αSMA in the CCl4-induced liver fibrosis model when compared with the normal rats (P < 0.01). 1: Normal group; 2: Model group.
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