Role Of Chemokine Receptor CXCR7 In Hepatocellular Carcinoma Growth And Its Mechanism

Hepatocellular carcinoma (HCC) occurs more frequently in China than in other countries all over the world, and is the third leading cause of cancer mortality. With undetected onset, long incubation period, high malignant degree and rapid development, great majority of patients were diagnosed with median or late-stage malignant tumors when they first came to visit a doctor. Frequent recurrence and a high incidence of metastasis have made the overall prognosis unsatisfactory and ultimately leads to death. Therefore, to clarify the molecular mechanism of HCC metastasis and find out biomarkers for clinical early and specific diagnose is very important for improving the living quality or survival rate of HCC patients.Our lab carried out comparative proteome analysis among similar genetic background but different metastatic potential human hepatoma cell lines, in order to characterize the molecular profiling of metastatic HCC. By a variety of proteomics technology and bio-informatics tools, a series of differentially expressed proteins were identified, and particularly the expression of CXCR7(chemokine receptor7) was apparently altered in HCC cell lines with metastatic potential. In the work of this dissertation, we tried to figure out whether the overexpression of CXCR7would have significant roles in the occurrence and development of primary HCC. Three parts of this dissertation are discussed as follows:Part I:Introduction. This chapter focuses on an overview of the mechanisms leading to tumor invasion and metastasis. Chemokines and their receptors have been proposed widely to have abnormal overexpression in many malignant tumors. Subsequently, detailed introduction of CXCL12and its receptor CXCR4confirms that tumor cells can take advantage of the interactions of chemokines and their specific receptors to organ-specific metastasis. Recently studies report that CXCL12also binds to chemokine receptor CXCR7with higher affinity than to CXCR4. Moreover, some groups suggest that elevated levels of CXCR7in tumors may play crucial roles in the pathogenesis and development of HCC. In addition, the construction process and metastatic characters of the HCC cell lines and animal models used in our work are described in this part.Part II:CXCR7expression in human hepatoma cell lines and clinical samples was investigated, and lentiviral expression vectors were constructed. The expression of CXCR7was evaluated in a panel of12kinds of HCC cell lines with different HCC metastatic potential, most of the non-metastatic HCC cell lines expressed low levels of CXCR7while CXCR7expression was gradually enhanced with increasing metastatic potential of HCC cell lines. We then evaluated CXCR7expression in10pairs of HCC specimens, compared to adjacent tissues of clinical specimens, CXCR7expression was hugely increased in tumor tissues. Lastly, we analyzed CXCR7protein expression profile in48HCC samples including24primary HCCs with metastasis and24HCCs without metastasis using immunohistochemistry. The expression level of CXCR7in HCC with metastasis was apparently higher than that in HCC without metastasis (P<0.001). This section we also constructed successfully lentiviral expressing vectors pLK0.1-CXCR7shRNAs which could interference CXCR7expression and pBabe-CXCR7which could up-regulate CXCR7expression. Later on, we would explore the effects of altered CXCR7expression on HCC development.Part III:Eftects of CXCR7expression on biological characteristics of human hepatoma cells. In vitro cell proliferation assays had shown that stable overexpression of CXCR7in HepG2or Hep3B cells led to accelerate cell proliferation, while stably transfected with a shRNA targeting CXCR7in LM3or97H cells suppress cell growth. Next we performed a cell cycle analysis and found that CXCR7depletion alters the cell cycle by arresting the Gl to S phase transition. Down-regulation of CXCR7expression could induce cell apoptosis, especially the early phase apoptosis using flow cytometry. This effect might be related with down-regulation of the expression of p-Akt and bcl-2whereas up-regulation of the expression of PARP-1. To investigate whether CXCR7accelerates tumorigenesis in vivo, we implanted subcutaneously with established stable cells and observed that increased exogenous CXCR7expression in HepG2cells generated larger tumors than control cells, whereas silencing CXCR7expression in LM3cells formed smaller tumors, statistically differed from those of vector control in both tumor size and weight. Therefore, we infer that CXCR7expression is critical for tumor growth and CXCR7may provide a venue to ameliorate tumor progression.The main contribution of this dissertation are as follows:CXCR7expression was closely correlated to metastatic status in HCC cell lines and clinical samples, and it was supposed to promote tumor growth. High expression of CXCR7increased cell proliferation of HCC cells in vitro. Overexpression of CXCR7in HepG2cells promoted HCC tumourigenesis in vivo, while subcutaneously implanted LM3cells transfected with CXCR7shRNAs showed tumor growth inhibition. However, CXCR7interacting proteins and regulating pathway remain unknown, we prefer tandem affinity purification (TAP) technology and protein chip strategy to reveal the molecular mechanisms of CXCR7in promoting tumor growth and HCC metastasis and explore the potential value of CXCR7as HCC diagnostic markers and drug targets.