61P The cysteine-rich protein 61 (Cyr61) contributes to tumor proliferation and invasion via HGF-mediated NF-kB signaling pathway in human hepatocellular carcinoma

CCN growth factor family regulates a variety of biological events such as cell proliferation, cell invasion, cell differentiation, apoptosis, and growth inhibition. Cysteine-rich protein 61 (Cyr61) is one of six secreted proteins in the CCNs. It has been shown that Cyr61 plays an important role in tumorigenesis and carcinogenesis in glioma, breast cancer, and gastric cancer, and others. However, Cyr61 has not yet been elucidated on the tumorigenesis, proliferation, and invasion of hepatocellular carcinoma, and needs to be researched and discovered. In this study, the HGF-mediated association of Cyr61, interleukin-8 (IL-8), and NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells) and cancer cell proliferation and invasion were investigated in two types of hepatoma cell lines. In this study, cell culture, cDNA microarray analysis, western blotting, Real-time Polymerase chain reaction, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Cyr61 knock-down with short hairpin RNA (shRNA), chromatin immunoprecipitation assay, Standard two chamber invasion assay. First, we confirmed that the expression level of Cyr61 was up-regulated by HGF (hepatocyte growth factor) in hepatoma cells. To identify associated pathway of HGF-induced Cyr61 about IL-8, NF-κB expression, the cells were treated with PI3K (Phosphoinositide 3-kinase) inhibitor (LY294002) and then analyzed by Western blotting. The HGF-mediated IL-8 and NF-κB levels were decreased with LY294002. The role for Cyr61 associated with IL-8 and NF-κB was determined by knock down cell of Cyr61. Cyr61-sh RNA cells showed a decreased level of IL-8 and NF-κB. HGF-mediated cell proliferation and invasion were decreased in Cyr61 knock down cell. These results suggest that Cry61 plays an important role in cell proliferation and metastasis in hepatocellular carcinoma, and Cry61 may be a novel target for the prevention of progression and treatment of hepatocellular carcinoma.