Cyr61/CCN1 Is Regulated By β-catenin In Human Hepatocellular Carcinoma

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. In this study, we observed and analysed expression of Cyr61 in a variety of human tumors firstly. Then, to study effect ofβ-catenin on Cyr61, hepatocellular carcinoma line HepG2 was infected with Adβ-catenin to overexpress exogenousβ-catenin, Adsi-β-catenin to knockdown of endogenousβ-catenin due to RNA interference-mediated gene silence, and AddnTcf4 to inhibit activity ofβ-catenin/TCF4 complex. Chromatin immunoprecipitation(CHIP) assay and luciferase reporter assay were used to study direct effect ofβ-catenin on Cyr61. 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. HE and Immunohistochemisty staining were used to detect expression of Cyr61 in multiple organs tumors tissue array and 30 human tumors cases (include tumors from esophagus,stomach,liver,colon, breast and lung).2. HepG2 cells were infected with Adβ-catenin and AdGFP, then Cyr61 was detected by RT-PCR next 2 to 4 days after infection.3. HepG2 cells were infected with Adsi-β-catenin and AdSES-hus, then Cyr61 was detected by RT-PCR and Western-blot next 2 to 4 days after infection.4. Exogenously expressed dnTcf4 was used to inhibit the transcriptional activity ofβ-catenin/TCF4 complex in HepG2 cells, then expression of Cyr61 was detected by Western-blot.5. CHIP assay was used in order to exploreβ-catenin's role in regulating expression of Cyr61 directly.6. A fragment, which contains two TBEs in the promoter of Cyr61, was amplified by PCR and constructed luciferase reporter recombinant plasmid with pGL3.7. Luciferase reporter assay was used to studyβ-catenin's role in regulating the promoter of Cyr61 directly.Results1. The expression of Cyr61 was found in human tumors tissue array and in human tumors cases with positive straining in 77﹪(38/48) and 93﹪(38/48) ,respectively. Strong positive staining was defined in tumors from brain, esophagus and thyroid. Positive staining was defined in tumors from stomach,liver,colon,rectum,lung,kedney,uterine,skin,ovary and breast. Negative staining was defined in tumors from prostate, bladder and pancreas.2. The expression ofβ-catenin increased significantly in the HepG2 infected with Adβ-catenin 48h after infection .Over-expression ofβ-catenin signaling increased expression of Cyr61 at mRNA level in HepG2 cell line.3. The expression ofβ-catenin decreased significant in the HepG2 infected with Adsi-β-catenin 48h after infection. RNAi mediated knockdown ofβ-catenin decreased endogenous Cyr61 mRNA and protein expression.4. The transcriptional activity ofβ-catenin/TCF4 complex was significantly inhibited by dnTcf4 after HepG2 was infected with AddnTcf4 for 30h. Then Cyr61 protein expression was decreased.5. Result of CHIP assay showed thatβ-catenin/Tcf4 signaling specifically binds to the Cyr61 promoter in vivo.6. Luciferase reporter assay showed thatβ-catenin signaling can regulate the promoter of Cyr61 directly.Conclusion1. Overexpression of Cyr61 was seen in many human tumors and the expression was different in different types of tumors. Cyr61 is probably used to diagnose the tumor as a new marker.2. Cyr61 is a new target gene of Wnt/β-catenin signaling in HepG2. Cyr61 overexpression is related to the abnormal canonical Wnt/β-catenin signal pathway in hepatocellular carcinoma and may participate in hepatocarcinogenesis and cancerometastasis.