| Objective Hypoxia, a typical feature of tumors, is involved in tumor pathogenesis and development. Hypoxia plays an important role in various functional responses in tumor cells, including apoptosis and angiogenesis. Aggressive tumor cells can mimic embryonic vasculogenic networks and form vasculogenic mimicry(VM). Our preliminary work demonstrated that Bcl-2/Twist1 co-overexpression can promote VM formation, but the underlying mechanism remains unclear. This study aimed at the mechanism of how Bcl-2 and Twist1 promoted VM formation.Methods 1. We compared the levels of Bcl-2 and Twist1 protein and m RNA expression in various HCC cell lines using Western blot, we found that Hep G2 had high Bcl-2 expression and low Twist1 expression in contrast to Bel7402, which presented low Bcl-2 expression and high Twist1 expression in the present study. Thus, we chose these two cell lines to demonstrate the importance of co-overexpression of Bcl-2 and Twist1. 2. Hep G2 and Bel7402 cells were treated Co Cl2, the expression of Bcl-2 and Twist1 proteins were measured using western blot. And the ability of VM formation before and after hypoxia was tested using 3D culture assay. 3. Hep G2-control, Hep G2-Twist1, Bel7402-control, and Bel7402-Bcl-2 cells were established using transfection. Stable transfected clones were selected and confirmed by western blot and immunofluorescence. The expression of EMT(epithelial-mesenchymal transition) markers, stem cell markers and endothelial markers were tested by western blot. 4. The Hep G2-control, Hep G2-Twist1, Bel7402-control, and Bel7402-Bcl-2 cells were then analyzed for functional changes in proliferation, invasion, migration, and clone formation ability using MTT, wound healing, invasion, clonogenic and 3D culture assay. 5. Hep G2 cell knockdown by si RNA decreased Bcl-2 expression, and Bel7402 cell knockdown by si RNA decreased Twist1 expression. Control and si RNA cellswere placed under hypoxic conditions. The protein expression levels of Bcl-2, Twist1, EMT markers, stem cell markers and endothelial markers were detected by western blot analysis for 0, 12, 24, 36, 48, and 60 h. 6. Incomplete femoral artery ligation was performed in the right hindlimbs of nude mice, the left hindlimbs weren’t ligated as control. Tumor cells were injected in both left and right hindlimbs and the effect of hypoxia on tumorigenesis abilities were observed. The expression of Bcl-2, Twist1, EMT markers, stem cell markers and endothelial markers in the engrafted tumor were tested by Immunohistochemistry(IHC) and the VM formation was tested by endomucin/PAS double staining.Results 1. Hypoxia could upregulate Bcl-2 and Twsit1 protein expression. Bcl-2 may assist Twist1 in entering the nucleus by initially associating with the pore structure of the nuclear membrane. The co-overexpression of Bcl-2 and Twist1 could promote VM formation. 2. Under normoxia, Bcl-2/Twist1 cooperation could also upregulate the protein expression of vimentin, stem cell markers and VM-associated markers and downregulate the expression of E-cadherin. The cell proliferation, migration, invasion, clone formation and VM formation abilities were also promoted(p<0.05). 3. Downregulation of Bcl-2 in Hep G2 cells and Twist1 in Bel7402 cells weakened the effects of hypoxia. Compared with control groups, hypoxia had less effects on the expression of EMT markers, stem cell markers and VM-associated markers of downregulation groups. Hypoxia promoted the VM formation of control groups, while downregulation groups couldn’t form VM before or after hypoxia. 4. In in vivo experiments, the engrafted tumors in the hypoxia group grew faster after the early hypoxia stage compared with the control group. Compared to the control groups, the protein expression of Bcl-2 in plasma, Twist1 in nucleus were higher in hypoxia groups(p<0.05). Moreover, the expression of vimentin, stem cell markers and VM-associated marker were higher(p<0.05) and the expressionof E-cadherin was lower(p<0.05) in the hypoxia groups compared with the control groups. The numbers of VM channels of hypoxia groups were also larger(p<0.05).Conclusions 1. Bcl-2/Twist1 co-overexpression and cooperation played an important role in VM formation. 2. Bcl-2/Twist1 cooperation promoted VM formation by inducing EMT and cancer cell stemness. |
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