| Background:Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors and the second leading cause of cancer-related death in China. Due to the high HBV prevalence, an estimated 0.78 million new liver cancer cases occurred worldwide, with China making up 50% of the total number. With long-term efforts, basic research and clinical treatment of liver cancer in China has made significant progress. However, the rate of recurrence within five year is up to 60% after liver resection, and the tumor recurrence after liver transplantation (LT) is also the main factor influencing patients’survival rate. Hypoxia is a common character of HCC. It can promote pro-survival reactions such as angiogenesis and tumor metastasis, finally, the malignant behavior of HCC could be elevated and the prognosis be reduced. Hereby, it will be important to illustrate the molecular mechanisms of HCC under hypoxic condition, and to establish new targets for therapeutic approach which can ameliorate the prognosis of HCC patients.Objectives:By Affymetrix Human U133 plus 2.0 array, the purpose of this research is to screen differentially expressed mRNA in normoxic and hypoxic HCC cells. Next, to seek the potential molecular biomarkers which are related with HCC development and recuurence under hypoxic condition through bioinformatics analysis. The potential molecular biomarker H2AX was selected, we firstly investigate the expression level of H2AX and its Phosphorylated products (y-H2AX) in HCC patients’tissues who underwent liver resection or LT. Then, we analyze the relationship between H2AX/γ-H2AX expression and clinicopathologic features and the prognosis of HCC patients with LT. Finally, we further study the biological behavior of H2AX/y-H2AX on HCC cell lines and illuminate its patential molecular mechanism in HCC.Methods:1. mRNA array hybridization was used to compare the mRNA expression profiles in normoxic and hypoxic cells. Data analysis selected many mRNA with expression that differed between normoxic and hypoxic cells at 2,8,24hours.2. RT-PCR was used to estimate the expression of H2AX in 32 samples of HCC tissue (After liver resection). Immunohistochemistry(IHC) was used to detect the levels of y-H2AX in 57 samples of HCC (After LT),37 samples of peritumoral tissue and 17 samples of normal tissue.3. According to the IHC results, we analyzed the relationship between H2AX/y-H2AX expression and clinicopathologic features and the prognosis of HCC patients with LT.4. HCC cell lines were stably transfected with specific knockdown lentivirus targeting H2AX. And then, cell counting kit-8 (CCK8) assay, transwell tumor invasion and migration assay and in vitro tube formation assay were used to detect the effect of H2AX on HCC cell proliferation, migration, invasion and angiogenesis. Nude mouse tumorigenicity assay was used to evaluate the effect of H2AX on tumorigenesis capability of HCC cells in vivo, and the experimental pulmonary metastasis model was used to investigate the effect of H2AX on invasion of HCC cells in vivo.5. Protein Chip technology was used to predict the downstream proteins which were regulated by H2AX/y-H2AX. Western blot,Immunofluorescence and Co-immunoprecipitation were used to explore the potential molecular mechanism of H2AX/y-H2AX in HCC development.Results:1. Data analysis selected H2AX mRNA with expression that differed between normoxic and hypoxic cells at 2,8,24hours. We confirmed H2AX expression change using RT-PCR, which showed H2AX to be significantly down-regulated in hypoxia culture.2. To investigate the level of H2AX in HCC patients, RT-PCR was used to estimate the expression of H2AX in 32 samples of HCC tissue. Unfortunately, tissue level of H2AX expression did not differ (p=0.3878);the levels of y-H2AX in 57 samples of HCC,37 samples of peritumoral tissue and 17 samples of normal tissue were examined by immunohistochemistry. The results showed that y-H2AX levels were higher in tumor tissues of patients than peritumoral tissues (p= 0.001) and normal tissues (p=0.001).3. All 57 patients with HCC after LT were divided into two groups:the high-expression group and low-expression group. Tumor size, vascular invasion and TNM stage showed significant differences (p< 0.05). Kaplan-Meier analysis revealed that HCC tissues with high expression of y-H2AX had either worse overall survival (p= 0.002) or shorter tumor-free survival (p< 0.001).4. Cellular experiments revealed that H2AX knockdown could inhibit the cell proliferation, cell invasion and migration, angiogenesis and EMT ability of HCC cells in vitro. In vivo animal experiment revealed that knockdown of H2AX could reduce angiogenesis and tumorigenicity of HCC, and could inhibit tumor metastasis in animal pulmonary metastasis model.5. The further molecular mechanism study revealed that y-H2AX is essential for HIF-la-mediated VEGF expression under hypoxic condition, H2AX knock-down could significantly inhibit the total protein expression of EGFR and EGFR-p845 in HCC cell lines under normoxic or hypoxic conditions, and H2AX knock-down could also effectively inhibited the EGFR translocating into the cell nucleus. EGFR siRNA significantly inhibited EGFR, HIF-la and VEGF protein expression levels but not y-H2AX level. These results suggest that EGFR and HIF-1α were the downstream protein of y-H2AX. y-H2AX is associated with HCC development through EGFR/HIF-la/VEGF signaling pathway. y-H2AX is a novel marker in the prognosis of HCC after LT and a potential therapeutic target.Conclusion:1. The expression levels of y-H2AX were higher in tumor tissues of patients than peritumoral tissues and normal tissues, and HCC tissues with high expression of y-H2AX had either worse overall survival or shorter tumor-free survival. y-H2AX is a novel marker in the prognosis of HCC after LT and a potential therapeutic target.2. There existed a y-H2AX/EGFR/HIF-1α/VEGF signaling pathway that could regulate tumor angiogenesis and metastasis under hypoxic condition in HCC development... |
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