The Role Of ZEB2 In Hepatocellular Carcinoma’s Vasculogenic Mimicry Formation

Background and Objective:Hepatocellular carcinoma(HCC) is one of the most prevalent primary malignancies with high incidence and mortality rates. Vasculogenic mimicry(VM),a unique microcirculation pattern, is closely related to tumor invasion,metastasis and tumor-related death. Although epithelial-mesenchymal transition(EMT) may be involved in VM formation, but the regulatory mechanism between EMT and VM is still not clear. This study aimed at clarifying the relationship between EMT and VM by TGF-β1 treatment in Hep G2 cells and further exploring the possible mechanism of EMT regulated by zinc finger E-box binding homeobox 2(ZEB2) on VM formation in HCC.Methods:1)IHC staining and PAS-CD34 dual staining were used to detect the expression of the ZEB2 and VM existence in 92 HCC tissue.2)Hep G2 cells was induced by TGF-β1 to observe morphologic changes and VM formation before and after induction.3)Expression of the transcription factors of Twist1, Snail, Slug, and ZEB2; EMT markers of E-cadherin and Vimentin and the expression of endothelial-associated proteins VE-cadherin were detected by Western blot analysis.4)The expression level of ZEB2 were evaluated in various HCC cell lines by using Western blott analysis.5)Three-dimensional culture(3D) was used to investigate the VM formation of ZEB2 up-regulation and knockdown cell lines in vitro, Western blot analysis was used to detect the expression of endothelial-associated proteins.6)Western blot analysis and immunofluorescent staining were used to detect the expression of EMT-associated proteins.7)Wound healing and invision assays were performed to detect the influence of ZEB2 on cell movement and invasion of HCC.8)Zymographic assays were performed to detect the activities of MMP-2 and MMP-9.Results:1)VM exists in HCC and the incidence is 18%. VM-positive HCC samples expressed higher ZEB2 than the VM-negative samples. The expression of ZEB2 was associated with VM formation. Multivariate analysis indicated that ZEB2 was an independent predictor for overall survival.2)TGF-β1-treated cells led to significant morphologic changes resembling mesenchymal phenotypes and could form VM in 3D culture.3)E-cadherin protein decreased and Vimentin protein increased after TGF-β1treatment. Of these transcription factors, ZEB2 has the most prominent increase in expression. VE-cadherin showed higher expression in TGF-β1-treated cells.4)Western blot analysis showed that Hep G2 had a low-level ZEB2 expression in contrast with the Bel7402, which presented a high-level ZEB2 expression.5)ZEB2 transfection of Hep G2 cells could formed typical pipe-like structures on the3 D Matrigel medium. An up-regulation of VE-cadherin,Flt and Flk expression based on the ZEB2 transfection of Hep G2 cells and a down-regulation of VE-cadherin,Flt and Flk expression on Bel7402-ZEB2 transfectant cells.6)ZEB2 up-regulation group presented an E-cadherin expression down-regulated and vimentin expression up-regulated and ZEB2 knockdown group presented increased E-cadherin expression and decreased vimentin expression.7)ZEB2 up-regulation in Hep G2 cells significantly promoted the ability of migration and invasion.8)ZEB2 up-regulation in Hep G2 cells inceased significantly the activities of MMP-2and MMP-9.Conclusions:1)ZEB2 associated with VM formation and metastasis and is an unfavorable prognostic indicator.2)In Hep G2 cells,TGF-β1could induce EMT and promote VM formatin by inceasing the expression of ZEB2 significantly.3)ZEB2 increased HCC cell invasion, migration, and VM formation in vitro.4)ZEB2 increased activities of MMP-2 and MMP-9 and expression of VE-cadherin,Flt-1, and Flk-1, thus promote VM formation.