Studies On The Correlation Between VPS4B And Hepatocellular Carcinoma

Objective 1. The study was designed to explore the expression of VPS4 B and its relationship with prognosis in patients with primary hepatocellular carcinoma patients, and investigate the significance of VPS4 B abnormal expression in the occurrence and development of hepatocellular carcinoma. 2. We investigated the expression variation of VPS4 B during the proliferation process of hepatocellular carcinoma cell line Huh7 and Hep G2 cells by serum starvation and refeeding experiment to explore the role of VPS4 B during the process. We aim to futher reveal the molecular pathogenesis of HCC, and to hope to provide a new target for the therapy of HCC.Methods 1. On histological level, in first, Western blot analysis was performed to determine the expression pattern of VPS4 B in 8 paired fresh human hepatocelluar carcinoma tissues and non-tumorous adjacent tissues. Meanwhile, immunohistochemical staining was used to detect the expression of VPS4 B and Ki-67(a proliferation index) in 98 cancerous liver samples,and statistical methods was used to determine the correlation between VPS4 B and Ki-67 expression with clinicalpathological characteristics,such as age, gender, AFP level, Hbs Ag, cirrhosis, histological grade, tumor size, metastasis and so on. Survival curve was calculated in patients with hepatocellular carcinoma using the Kaplan-Meier analysis, and the prognosis was analyzed by the univariate and Cox proportional hazards model. 2. On cellular level, Hu H7 and Hep G2 cells as common cells that displayed with high VPS4 B expression were selected as the research object, and they were treated with serum starvation and release for synchronization and then the cell cycles were detected by flow cytometer. Western blot was performed to detect the expression of VPS4 B, cyclin A and PCNA in Huh7 and Hep G2 cells during serum starvation and releasing process. After the knockdown of VPS4 B expression in HCC cells by small interfering RNAs, flow cytometry analysis and Western blot were adopted respectively to determine cell cycle progression and cell proliferation.Results 1. Both Western blot and immunohistochemical analysis revealed that VPS4 B expression was significantly upregulated in hepatocellular carcinoma compared with adjacent nontumorous liver tissues or in poorly differentiated specimens compared with well-differentiated ones. The expression of VPS4 B was significantly associated with histological grade(P=0.026) and vascular invasion(P=0.033), and correlation analysis indicated that there was a positive correlation between VPS4 B expression and Ki-67-based proliferative activity(r2=0.398;P=0.003); The Kaplan-Meier survival curves revealed that high VPS4 B expression was correlated with a poor survival with statistical significance(P<0.01). Unvaried analysis indicated that VPS4 B expression was associated with poor prognosis of hepatocellular carcinoma(P=0.001). Multivariate Cox proportional hazards regression analysis showed that VPS4 B expression was an independent prognostic marker for hepatocellular carcinoma(P=0.016). 2. The cell cycle of Hu H7 and Hep G2 cells were blocked by serum starvation, but the HCC cells were reentering the cell cycle after serum addition. Flow cytometry analysis revealed that the HCC cells were released from the G1 phase and they reentered the S phase respectively upon serum refeeding. Furthermore, Western blots also showed that the expression of VPS4 B, cyclin A and PCNA was progressively increased after serum-refeeding in HCC cells during cell cycle progression as expected. si RNA analysis showed that VPS4 B depletion could inhibite cell proliferation and resulted in cell cycle arrest.Conclusions 1. The expression of VPS4 B was significantly up-regulated in hepatocellular carcinoma and might serve as an independent prognostic marker for hepatocellular carcinoma patients,which suggested that VPS4 B might play a role in the oncogenesis and development of hepatocellular carcinoma. 2. During the proliferation process of Huh7 and Hep G2 cells, the expression of VPS4 B was up-regulated. After VPS4 B was down-regulated by si RNA, the growth of hepatocellular cells was inhibited, which suggested that VPS4 B may influence the HCC cell cycle progress and proliferation. 3. Owing to overexpression of VPS4 B in hepatocellular carcinoma and its important role in cell cycle and cell proliferation,VPS4 B might serve as a novel candidate gene for the diagnosis and therapeutic strategies for HCC in future.