The Research Of Molecular Mechanism For The Role Of Mig/CXCR3 In Transendothelial Migration Of Hepatocellular Carcinoma Cells In HBV-related HCC

Background and Objective Hematogenous metastasis is an early event in hepatocellular carcinoma progression and the underlying mechanisms are still unclear. Metastasis is a multi-step cascade process in which transendothelial migration(TEM) of cancer cells into vessels is one of the rate-limiting procedures. It is known now that not all the cancer cells own the metastatic characteristics. Previous studies have demonstrated that cancer stem cells(CSCs) are proposed to initiate cancer propagation and metastasis, and CD133 is a putative HCC cancer stem-cell marker. There is increasing evidence that tumor microenvironment(including chemokine) plays a pivotal role in the dissemination and establishment of CSCs metastasis. For proof of principle, we have illustrated that Mig protein expressed higher in hepatitis B-related HCC tissues than that in normal liver counterparts. In addition, the level of CXCR3 expression was positively correlated with HCC metastasis. Further, it was shown that stimulation with chemokine Mig lead to profound migration of the CD133positive(CD133+) hepatocellular carcinoma cells through endothelial monolayer as compared with the control group. Yet the molecular mechanisms for tumor cells metastasis promoted by chemokine Mig have so far remained obscure. In the current proposed project, we will try to investigate the impact of Mig/CXCR3 in CD133+ hepatocellular carcinoma cells and endothelial cells in vitro, and to clarify the effect of the pathway on hepatoma recurrence and metastasis in vivo. Then discover potential targets for early anti-metastasis intervention in hepatoma.Methods Using Collagenase perfusion method extracts umbilical vein endothelial cells, then investigate the influence of soluble Mig on interendothelial molecules by RT-PCR ?western blot and immunohistochemistry. In addition, we use CCK-8 assay to testify the impact of soluble Mig on umbilical vein endothelial cells proliferation. Moreover, the CD133+ cells were sorted by Flow cytometry, then they co-cultivated with umbilical vein endothelial cells. After treated with soluble Mig, the invasion migration and adhesion of CD133+ cells were investigated by transwell assay and adhesion assay; And the migration of umbilical vein endothelial cells were also tested by transwell assay.Result We found that Mig is able to promote cell–cell disruption in endothelial monolayers, which could be due to decreasing endothelial cells proliferation or reducing the expression of gap junction protein Cx32 and adhesion molecule VE-cadherin. Interestingly, when human umbilical vein endothelial cell monolayers were stimulated or not stimulated with Mig and overlaid with CD133+ cells for 24 h, the migration ?invasion and adhesion of CD133+ cells were increased;Meanwhile, the migration of human umbilical vein cells were also improved, and the expression of gap junction protein Cx32 was reduced.Conclusion Stimulation with chemokine Mig lead to profound migration of the CD133+ hepatocellular carcinoma cells through endothelial monolayer as compared with the control group. The molecular mechanisms for tumor cells metastasis promoted by chemokine Mig may were decreasing endothelial cells proliferation or reducing the expression of Cx32 and VE-cadherin. Those evidences may clarify the role of Mig/CXCR3 on CD133+ liver cancer cell migration across endothelial barrier and its molecular mechanism.