| 1. BackgroundHepatocellular carcinoma (HCC) which is a primary malignancy arising within the liver, is the fifth most frequent cancer worldwide and the third leading cause of cancer mortality. Genetic alterations and epigenetic high-risk factors, such as chronic infection with HBV or HCV, hepatic cirrhosis, alcoholic liver disease and aflatoxins, account for the high morbidity of HCC.HCC often arises within a diseased liver and represents a lethal complication of cirrhosis. The poor prognosis of HCC patients reflects the lack of understanding of liver carcinogenesis and the failure to develop interventions that are aimed at blocking or reversing the steps of malignant transformation. Molecular targeted therapies focused on critical pathways are needed. Indeed, global gene expression profile studies have revealed the activation of several pathways that drive uncontrolled growth and aberrant survival signaling. Several key pathways have been identified. However, no novel gene markers or molecular prognostic factors have entered into clinical practice, emphasizing the need for new markers for diagnosis and prognosis of HCC patients.The abrogation of cell cycle checkpoints is an important hallmark that might promote hepatocellular cancer formation. As a cell cycle checkpoint regulator, cell division cycle20(Cdc20) and another regulate factor, Cdhl, directly bind to the anaphase-promoting complex/cyclosome (APC/C) and activate its cyclin ubiquitination activity, which is required for anaphase initiation and exit from mitosis. Recently, accumulating evidence showed that Cdc20may function as an oncogene, and was highly expressed in various types of human tumors, such as pancreatic ductal adenocarcinoma, breast cancer, glioblastomas, ovarian cancer and gastric cancer, etc. In line with these results, high expression of Cdc20was tightly associated with a poor prognosis of some tumors. However, the expression pattern of Cdc20in HCC and its association with clinical factors and the prognosis of HCC patients have not been well researched and remain largely unknown.2. Purpose To detect the expression level of Cdc20in hepatocellular carcinoma and investigate its role in progression and prognosis of HCC patients.3. Methods1) Bioinformatics analysis Analyzing the microarray dataset GSE14520from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) by using bioinformatics methods, and screening the differentially expressed genes between HCC and adjacent normal liver tissues. Then clusting these gene functions according to Gene Ontology categaries and constructing the molecular interaction networks. The target gene which plays an important role in HCC will be selected for further research.2) Cdc20expression in HCC tissues Quantitative real-time PCR and western blot analyses were used to examining the mRNA and protein level of Cdc20in16fresh HCC and adjacent normal liver tissues.3) Relationship between Cdc20and clinicopathological parameters and prognosis of HCC patients Immunohistochemistry (IHC) was used to detect the expression level and sites in132paraffin-embedded, archived HCC and adjacent non-tumor tissues. The relationship between Cdc20and clinicopathological parameters and prognosis of HCC patients were also analyzed.4) Suppressing Cdc20expression using siRNA and detecting the influence on cell proliferation, cell migration, cell cycle and downstream substrateSpecific siRNA was synthesized and transfected into HepG2cells to silence the Cdc20expression. Cellular proliferation assay, wound healing assay, and Fluorescence-activated cell sorting cell cycle analysis were adopted to observe the changes in cell proliferation, cell migration and cell cycle after gene silencing. Then detecting the expression change of the downstream substrate p21by using western blot.4. Results1) Cdc20is the major node in HCC molecular interaction networksBy using bioinformatics methods,137differentially expressed genes were selected. Among the molecular networks constructed with the137genes, Cdc20was the major node which interacted with several genes. Thus we speculated that Cdc20may play a key role in the development of HCC.2) Cdc20is overexpressed in HCC tissuesQuantitative real-time PCR and western blot analyses both revealed that Cdc20was up-regulated in14of16fresh HCC tissues compared with adjacent normal liver tissues.3) Overexpression of Cdc20was related with several clinicopathological parametersBy IHC staining, CDC20staining was significantly increased in HCC samples when compared to adjacent non-tumor samples (P=0.000), and the positive staining was mainly localized in the cytoplasm and nucleus. In addition, CDC20staining score was correlated with tumor differentiation (P=0.002) and TNM stage (P=0.022).4) Cdc20could be a biomarker of poor prognosis in HCC patientsAfter surgery, overall survival was observed for75of the132HCC patients, and the cumulative5-year survival rate was21%. Cox-regression univariate analysis, CDC20expression score (P=0.011) and certain HCC clinical factors such as tumor size (P=0.000), intra/extrahepatic metastasis (P=0.006), AFP level (P=0.032), tumor differentiation (P=0.000), TNM stage (P=0.003), p53expression (P=0.02) and Ki-67expression (P=0.004) demonstrated statistically significant associations with survival time. However, by using multivariate analysis we found that only the tumor size (P=0.000), tumor differentiation (P=0.000) and p53expression level (P=0.000) were demonstrated as the independent predictive factors for poor outcome of HCC.5) Suppression of Cdc20expression by siRNA leading to reduced cell growth, cell migration and increased number of cells in the G2/M phaseCell proliferation assays revealed that the growth of Cdc20siRNA-transfected cells was remarkably inhibited when compared to cells transfected with the negative control siRNA. An increased number of cells in the G2/M phase was also observed in siCDC20-transfected cells. Wound healing assay revealed that cell migration was not significantly affected after Cdc20expression suppressed. Examining the downstream substrate p21protein level we found that the p21protein level was remarkably up-regulated in response to the suppression of CDC20.5. Conclusions1) Cdc20is overexpressed in HCC.2) Overexpression of Cdc20is correlated with the tumor differentiation and TNM stage of HCC.3) Up-regulation of Cdc20has relevance to the poor prognosis of HCC patients.4) Suppressing Cdc20expression may result in a suppressive effect on liver cancer cell proliferation and induce cell cycle arresting in G2/M phase. Cyclin-dependent kinase inhibitor1A (p21) protein level was remarkably up-regulated in response to the suppression of CDC20. |
