Matrix stiffness promotes epithelial-mesenchymal transition and vasculogenic mimicry formation through YAP in glioma cells

Abstract

Objective To investigate the mechanism of matrix stiffness promoting epithelial-mesenchymal transformation (EMT) and vasculogenic mimicry (VM) of glioma cells by yes-related proteins (YAP). Methods Human glioma U87 cell lines were cultured under different matrix stiffness conditions. Matrix stiffness 0.2, 16.0 and 64.0 kPa represented normal brain, glioma and extreme stiffness, respectively. CCK-assay was used to examine the proliferation of U87 cell line.The localization of YAP cells was detected by immunofluorescence staining. The expression of YAP targeting genes including CTGF and CRY61 were detected by real-time quantitative PCR. The expression and phosphorylation expression of YAP, proliferation signal including Akt and ERK1/2 were detected by Western blot. Immunofluorescence and Western blot were used to detect the expression of EMT-related proteins including Vimentin, E-cadherin and Twist. VM formation was detected in soft and stiff substrates under 3D culture. U87 cells were transfected with shYAP lentivirus, and then the expression of EMT proteins and VM formation were examined. Results With the increase of matrix stiffness, the proliferation activity of glioma cells and phosphorylation expression of Akt and ERK1/2 were increased (all P<0.05), the expression of phosphorylated Yap was decreased (P<0.05), while the expression levels of CTGF and CRY61 were increased (both P<0.05) as the nuclear translocation of YAP was increased. The results of immunofluorescence staining and Western blot showed that with the increase of matrix stiffness, the EMT component expression levels of Vimentin and Twist were increased, the epithelial marker E-cadherin was decreased (all P<0.05). Compared with that under soft matrix 3D culture, the formation of VM tubular structure in U87 cells was increased in stiff matrix (P<0.05). Interference with Yap expression inhibited the enhancement of the proliferation of glioma cells, the expression of Vimentin and Twist, and VM formation induced by matrix stiffness (all P<0.05). Conclusions Matrix stiffness promotes the proliferation of glioma cells, EMT and VM formation through YAP. Targeted matrix stiffness and YAP can be used as new strategies for glioma treatment. Key words: Glioma; Matrix stiffness; Yes-associated protein; Epithelial-mesenchymal transition; Vasculogenic mimicry