| ObjectiveThe aberrant activation of the canonical Wnt/β-catenin signal pathway is evident in embryonic developmental anomaly and tumorigenesis.β-catenin, a key mollecullar in canonical Wnt signaling pathway,overexpression is relative to hepatocarcinogenesis. The cysteine-rich angiogenic protein 61 (Cyr61) is an extracellular matrix-associated protein that mediates extracellular matrix development and cell proliferation, differentiation and adhesion, stimulates cell migration, augments growth factor–induced DNA synthesis, enhances cell survival and angiogenesis. Much attention has been paid to the relation between Cyr61 and tumergenesis, but what is the mechanism is still not clear, especially concerning hepatocarcinogenesis. Expressions ofβ-catenin and Cyr61 were studied in hepatoma carcinoma cell lines and hepatocellular carcinoma (HCC) tissues. We found that abnormal expression of Cyr61 significantly related with abnormal expression ofβ-catenin. Then, to study effect ofβ-catenin on Cyr61, hepatocellular carcinoma line HepG2 was infected with Adβ-catenin to overexpress exogenousβ-catenin and Adsi-β-catenin to knockdown of endogenousβ-catenin due to RNA interference-mediated gene silence. This study established the foundation for further studies on the function and mechanism of Cyr61 in tumorigenesis, and provided a new tumor marker and a gene therapy target for tumor diagnosis and therapy.Methods1. Expressions ofβ-catenin and Cyr61 were studied by immunohistochemistry and RT-PCR in hepatoma carcinoma cell lines HepG2, QGY-7701 and H22 and hepatocyte L02.2. Protein expressions ofβ-catenin and Cyr61 were studied by immunohistochemistry in 62 patients. The relationship ofβ-catenin and Cyr61 expressions with clinical pathological features was evaluated. The relativity betweenβ-catenin and Cyr61 was also assessed.3.β-catenin mRNA and Cyr61 mRNA were detected by RT-PCR in 30 patients. The relativity betweenβ-catenin and Cyr61 was also analysed.. 4. Recombinant adenovirus vectors Adβ-catenin (with GFP labeling), AdGFP (as control) and Adsi-β-catenin infected HEK293 cells respectively for amplifying these virus. Construct and amplify recombinant adenovirus vector AdSES-hus(as control).5. HepG2 was infected with Adβ-catenin and AdGFP, then Cyr61 was detected by RT-PCR next 3 days after infection.6. HepG2 was infected with Adsi-β-catenin and AdSES-hus, then Cyr61 was detected by RT-PCR next 2 to 4 days after infection.Results1. Expressions of Cyr61 andβ-catenin at both mRNA and protein levels were found in hepatocyte L02 and hepatoma carcinoma cell lines HepG2, QGY-7701 and H22, co-expression of Cyr61 andβ-catenin were 100% of all cells.2. The abnormal expressions ofβ-catenin protein and Cyr61 protein were found in 84.85% (53/62) and 77.42% (48/62) of 62 patients. The expression ofβ-catenin was higher in HCC tissues with vein infiltrating, cancerometastasis, multinodular and higher expression of AFP than in that with no vein infiltrating, cancerometastasis, mononodular and lower expression of AFP (P<0.05). The expression of Cyr61 was higher in HCC tissue with vein infiltrating, cancerometastasis, and multinodular than in that with no vein infiltrating, cancerometastasis, and mononodular (P<0.05). Bothβ-catenin protein and Cyr61 protein normal and abnormal expressed were detected in 72.58% (45/62) and 10% (6/62) respectively. Expression of Cyr61 protein was positively correlated with the expression ofβ-catenin protein (r=0.435, P<0.05).3. The expressions ofβ-catenin mRNA and Cyr61 mRNA were found in 100% of 30 patients. Expression of Cyr61 mRNA was positively correlated with the expression ofβ-catenin mRNA (r=0.856, P<0.01), according to the results of semi- quantity RT-PCR.4. The expression ofβ-catenin increased significantly in the HepG2 infected with Adβ-catenin 24h after infection, while the expression of Cyr61 also incrased in these cells at the same time.5. The expression ofβ-catenin decreased significant in the HepG2 infected with Adsi-β-catenin 48h after infection, while the expression Cyr61 also decrased in these cells at the same time. Conclusion1. Expressions ofβ-catenin and Cyr61 were found both in hepatoma carcinoma cell lines and hepatocellular carcinoma tissues, with highly co-expression ofβ-catenin and Cyr61. Expression of Cyr61 was related with expression ofβ-catenin, they may participate in hepatocarcinogenesis and cancerometastasis.2. Expression of Cyr61 increased significantly in HepG2 cell line that was overexpressed exogenousβ-catenin, and decreased significantly in HepG2 cell line that was inhibited endogenousβ-catenin expression. Expression of Cyr61 was related with expression ofβ-catenin, and the canonical Wnt/β-catenin signal pathway may directly or indirectly regulate the potential down-stream target gene Cyr61.Conclusively, Cyr61 overexpression is related to the abnormal canonical Wnt/β-catenin signal pathway in hepatocellular carcinoma. They may participate in hepatocarcinogenesis and cancerometastasis, and Cyr61 is maybe the target gene of Wnt/β-catenin signal pathway.
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