Epithelial-mesenchymal Transition Contributes To Malignant Biological Beheviour Of Hepatocellular Carcinoma:a Study Of Its Functional Role And Mechanisms

Hepatocellular carcinoma(HCC) is a common cancer and ranks the third mortality in China. Traditional therapies are insufficient for HCC. As the development of HCC is a complicate process with multi-genes and multi-pathways, the only way to cure HCC is uncover the mechanisms of it.The potency of invasion and metastasis is one of the most malignant biological behaviors of HCC. Most of patients suffering from HCC have no chance to receive surgical therapy because of metastasis. A large part of the patients who succeed to receive surgery suffer recurrence or metastasis. So, the metastasis, instead of primary tumors, account for the main cause of death. In addition, chemotherapy, including transcatheter arterial chemoembolization (TACE) and target medicine, is an important part of multidisciplinary therapy contributing to improved prognosis of HCC patients. However, many HCC patitents develop primary or acquired chemoresistance which lead to improvement of therapy. Some studies reveal that the residue cells after chemotherapy acquired higher potency of metastasis and malignancy. So it is urgent for us to explore the mechanism of metastasis and chemoresistance and to find effective intervention.The epithelial-mesenchymal transition (EMT) is a process which allows polarized epithelial cells to acquire mesenchymal properties including fibroblastoid morphology, characteristic gene express changes, increasing potential for motility, and in the case of cancer, increased invasion, metastasis, resistance to chemotherapy. The critical role of some transcriptional factors (TFs) in the activation of EMT has been well documented. These TFs include the Gsc, Snai1, Slug, Twist1, FOXC, FOXC2, Zeb1and Zeb2. On base of the above reasons, our present study aimed to explore the involvement of EMT in metastasis and chemoresistace of HCC from view of the EMT-related TFs. In part Ⅰ of this study, we initially assessed by comparing the expression profiles of known EMT-related TFs between HCC tissues with and without microvascular invasion (MVI), and found that FOXC1was the TF which presented prominently higher level in HCC tissues with MVI. We then explored the functional role of FOXC1in regulating EMT, cell proliferation, metastatic potential, and VEGF-A expression for further understanding the mechanism underlying MVI formation of HCC. In part Ⅱ of this study, we established the TACE model in vivo through treatment of cisplatin (DDP) in addition to hypoxia. We found the residue cells after chemotherapy and hypoxia developed EMT process. The express level of ZEB1in the residue cells increased significantly, implying the possible role of ZEB1in the formation of chemoresistance and resistance to hypoxia. We further explore the mechanism by which ZEB1contribute to chemoresistance and resistance to hypoxia. To our knowledge, our study is the first one to explore the involvement of EMT in regulating malignant biological behaviors of HCC, including metastasis and chemoresistance, et al. Part Ⅰ FOXC1Contributes To Metastasis and Invasion In Primary Hepatocellular Carcinoma Via Regulating Epithelial-Mesenchymal Transition.Aim:The existence of microvascular invasion (MVI) formation is one of the most important risk factors predicting poor outcome in hepatocellular carcinoma (HCC) and its mechanism remains largely unknown. Epithelial-Mesenchymal Transition (EMT) has been suggested to be involved in many steps of the invasion-metastasis cascade. To elucidate the possible contribution of EMT to MVI, we initially evaluated the expression of8EMT-related transcription factors (TFs) in HCC patients with or without MVI and found that FOXC1expression was significantly higher in patients with MVI than those without MVI, we further explored the possible role of FOXCI in regulating metastasis and invasion.Methods:15pairs of patients suffering from HCC were enrolled in this study, each pair consists of a patient with MVI and one without MVI, we used realtime RT-PCR to compared the expression of8EMT-related transcription factors (TFs) in HCC patients; we further used western-blot to compared the different expression of FOXC1in8pair of HCC tissues including tumor and non-tumor tissue and analysis the relationship between FOXC1and clinicopathological variable; Knockdown strategy in HCC cells Bel-7402and SK-Hep1was used to explore FOXC1role. Results:FOXC1expression was significantly higher in patients with MVI than those without MVI; FOXC1expression was related to MVI formation and differentiated level of in HCC patients; Knockdown of FOXC1expression in HCC cells resulted in a partial conversion of their EMT progresses, mainly regulating the mesenchymal component. Ectopic expression of snail, twist or TGF-β1could induce expression of FOXC1, but none of the expression of snail, twist, slug or TGF-β was consistently down-regulated in response to FOXC1silencing, suggesting FOXC1might operate the downstream of other EMT regulators. In addition, knockdown of FOXC1expression led to cytoskeleton modification accompanied by decreased ability of cell proliferation, migration, and invasion. Meanwhile, some matrix metalloproteinase.(MMPs) and VEGF-A were also simultaneously down-regulated.Conclusion:Our findings demonstrate that FOXC1is one of candidate predictive markers of MVI, and that inhibition of FOXC1expression can partially reverse EMT program, offering a potential molecular therapeutic target for reducing tumor metastasis in HCC patients Part II The study of mechanism by which Epithelial-Mesenchymal Transition Contributes To ChemoresistanceAim:Transcatheter arterial chemoembolization (TACE) is one of most important treatment option for advanced HCC, However, the effect of TACE on improvement of long-time survival is still in large debate, and TACE is even suspected to add the possibility of metastasis. The residue cells after chemotherapy and hypoxia display resistance to chemotherapy and hypoxia, which was thought to limit the application of TACE. The mechanism of cancer chemoresistance is of great concern both in clinic field and basic research. Recent researches propose the possible involvment of Epithelial-mesenchymaltransition(EMT) in chemoresistance formation.Methods:we established the TACE model, in vitro, through administration of DDP and hypoxia, these treatments were repeated3times, the residue cells were named DDP-,hypoxia-induced residue cells(DHR) including7402/DHR and LM3/DHR according to their original cells. EMT characteristic of EMT was evaluated through morphological change and transition of EMT-related gene. The tumor stem cell property was studied through clone formation, In Vivo Tumorigenicity and assess of the cell surface expression of some known markers. The ZEB1contribution to resistance to chemotherapy and hypoxia was studied using cck-8test; we tranfected ZEB1into HCC cells with lentivirus and established stable cell, realtime RT-PCR and western-blot were used to analysis the expression level of miR-200c in respond to ZEB1alternation, then the Luciferase Reporter Assay System was used to confirm the effect of ZEB1on the expression of miR-200c. In addition, we explored the role of miR-200c in regulating the expression of Bmi-1by using western-blot and Dual Luciferase Reporter Assay System.Results:7402/DHR and LM3/DHR resulted in morphological changes consistent with EMT including loss of cell polarity and fibroblast-like spindle-shaped morphology.7402/DHR and LM3/DHR displayed cancer stem cell features, such as increased ability of clone formation and tumorigenicity. In addition,7402/DHR and LM3/DHR showed moderate increased expression of CD133and CD326, while no different portion of CD44+CD24-cells was observed. These changes were associated with the alterative expression of EMT markers at a protein level. For example, expression of E-cadherin is decreased, the expression N-cadherin, Vimentin was increased. Among the EMT-inducing transcription factors (TFs), the expression level of ZEB1was significantly enhanced in response to cisplatin and hypoxia. The chemoresistance could be partly reversed through depressed ZEB1, implying its involvement in to chemoresistance. However, inhibition of ZEB1had no effect on resistance to hypoxia. Our results also showed decreased expression of miR-200c and miR-203in both7402/DHR and LM3/DHR cells, and the transfection of miR-200c could reverse partly the chemoresistance. Furthermore, ZEB1could suppress activity of the putative miR-200c promoter, indicating expression of miR-200c can be directly suppressed by ZEB1. Bmi-1was a candidate target of miR-200family, our western-blot and Dual Luciferase Reporter Assay System showed miR-200c could directly regulate the expression of Bmi-1.Conclusion:The residue cells after chemotherapy and hypoxia underwent EMT process and displayed some property of cancer stem cell; increased expression of ZEB1and decreased expression of miR-200c contribute to chemoresistance of HCC; ZEB1could directly suppress expression of miR-200c through regulating activity of the putative miR-203promoter.