Clinical Significance And Molecular Mechanism Of Hypoxia Inducible Factor 2α In Hepatocellular Carcinoma

Objective1. To investigate the expression, diagnostic and prognostic significance of hypoxia inducible factor(HIF-2α) in hepatocellular carcinoma(HCC). 2. To detect the expression of HIF-2α m RNA and protein in human HCC cell lines include Hep3B、HCCLM3、MHCC-97H、Hep G2、Huh-7 and human liver cell line HL-7702 under normal cell culture and Co C l2 simulated anoxia culture condition. And choose the most suitable cell line can be used to do next work. 3. To investigate the biological function change of HCC cells after lentivirus contained si RNA targeting HIF-2α infection. 4. To explore the potential molecular mechanisms of HIF-2α in HCC by genechip.Methods1. A total of HCC tissues from 90 patients who underwent curative surgery and with detailed clinicopathologic and follow-up data were collected. Immunohistochemistry was performed to detect HIF-2α 、 VEGF and CD34 expression in HCC tissues. ELISA was used to detect serum HIF-2α in 90 HCC patients and 20 healthy volunteers. Receiver operating characteristics was analyzed to calculate diagnostic accuracy of serum HIF-2α, alpha- fetoprotein(AFP) and their combination. 2. Real time quantitative PCR and Western blot were used to detect the level of HIF-2α m RNA and protein of the human HCC cell lines Hep3 B 、HCCLM3、MHCC-97H、 Hep G2、Huh-7 and human liver cell line HL-7702 which were cultured in the condition of normal and Co Cl2 simulated anoxia. 3. Lentivirus contained si RNA targeting HIF-2α were designed and synthesized and then infect Hep3 B cell line which was selected. The interference efficiency of RNAi was detected by RT-PCR. MTT assay was used to detect the ability of liver cell proliferation, and Annexin V-APC flow cytometry was used to detect the apoptosis of cancer cells. 4. Affymetrix gene chip was used to detect the alteration of gene expression after HIF-2α gene down-regulated, and bioinformatics was used to analysize the data.Results1. Immunoreactivity of HIF-2α was detected in 76.7% of HCC, and was significantly associated with microvascular invasion, pathological grade, tumor capsula status, TNM stage and positively correlated with VEGF(p < 0.001) and microvessel density(p < 0.001). The combination of serum HIF-2α and AFP had a markedly higher area under the curve than HIF-2α alone. HIF-2α expression was associated with unfavorable overall survival(OS)(p < 0.001) and disease- free survival(DFS)(p < 0.001). Multivariate analysis indicated that patients with HIF-2α-expressing tumors had a significantly shorter OS(p = 0.030) and DFS(p = 0.005) than those with HIF-2α-negative tumors. Thus, HIF-2α expression possibly plays an important role in tumor angiogenesis, progression and prognosis of HCC. Serum HIF-2α may serve as a biomarker for early HCC diagnosing and predicting outcome. 2. There was no significant difference of HIF-2α m RN A in all the detected cell lines under normal and Co Cl2 simulated anoxia culture condition. While the level of HIF-2α protein in most cell lines increased in varying degrees in the condition of Co Cl2 simulated anoxia culture. And Hep3 B was chose to do next work. 3. Lentivirus contained si RNA targeting HIF-2α infect the Hep3 B cell line successfully. Real time quantitative PCR showed HIF-2α gene was significantly down-regulated. MTT showed ability of cell pro liferation of Hep3 B cell line with HIF-2α down-regulated was significantly decreased, while the apoptosis cell number significantly increased compared with controls. 4. Gene chip assay revealed that there were 1352 genes expression altered after HIF-2α gene down-regulation. Among the 1352 genes, up-regulated were 674 genes and down-regulated were 678 genes. These genes are involved in 30 gene set include apoptosis, MAPK signal pathway, Wnt signal pathway and so on.Conclusion1. HIF-2α was positive expressed in HCC tissues and was significantly associated with microvascular invasion, pathological grade, tumor capsula status, TNM stage and positively correlated with tumor angiogenesis. It possibly plays an important role in tumor angiogenesis, progression and prognosis of HCC. HIF-2α may serve as a biomarker for early HCC diagnosing and predicting outcome. 2. There was no significant difference of HIF-2α m RNA in all the human HCC cell lines and human liver cell line under nomal and Co Cl2 simulated anoxia culture condition. While the level of HIF-2α protein in most cell lines increased in var ying degrees in the condition of Co C l2 simulated anoxia culture. Hep3 B was selected to do next work. 3. RNAi can efficiently down-regulate the HIF-2α gene in HCC cell line Hep3 B. The ability of cell pro liferation of Hep3 B can be inhibited by HIF-2α silencing, while cell apoptosis number significantly increased. 4. Many genes had a significantly differential expression after HIF-2α gene down-regulation. These genes are involved in several important signal transduct pathways such as MAPK signal pathway, Wnt signal pathway, cell apoptosis pathway and so on. However, it is still far from to reveal the HIF-2α molecular mechanism and signal pathways in HCC in this study. More researches are needed to carry out.