基质硬度通过YAP促进脑胶质瘤细胞上皮-间质转化和血管生成拟态形成的实验研究

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

目的探讨基质硬度通过yes相关蛋白(YAP)促进脑胶质瘤细胞上皮间质转化(EMT)和血管生成拟态(VM)形成的机制。方法在不同基质硬度条件下培养人脑胶质瘤U87细胞株,0.2、16.0和64.0 kPa 3种硬度分别代表正常大脑、胶质瘤和极度僵硬。以CCK8实验检测胶质瘤细胞的增殖能力,采用免疫荧光染色检测YAP细胞定位的改变,以实时定量PCR检测YAP靶基因CTGF、CRY61的表达,以蛋白印迹实验检测YAP及细胞增殖信号Akt、ERK 1/2的磷酸化表达。通过免疫荧光染色和蛋白印迹实验检测EMT相关蛋白Vimentin、E-cadherin、Twist的表达。在软、硬基质3D培养下检测VM形成的情况。转染shYAP慢病毒后,检测YAP干扰对基质硬度作用于胶质瘤细胞的增殖、间质蛋白(Vimentin和Twist)表达和VM形成的影响。结果随着基质硬度的增高,胶质瘤细胞的增殖活性及其增殖信号通路Akt、ERK 1/2的磷酸化表达增加(均P<0.05),胶质瘤细胞的YAP核转位增加,磷酸化YAP的表达逐渐下降(P<0.05),而CTGF和CRY61基因的表达均升高(均P<0.05)。免疫荧光染色和蛋白印迹实验结果表明,随着基质硬度的增高,EMT元件Vimentin、Twist的表达增高,上皮标志E-cadherin的表达减低(均P<0.05)。与软基质3D培养下相比,硬基质培养条件下U87细胞VM管样结构的形成增多(P<0.05)。干扰YAP的表达可减低较高的基质硬度促进胶质瘤细胞增殖、Vimentin和Twist的表达以及VM形成的作用(均P<0.05)。结论基质硬度可通过YAP促进胶质瘤细胞的增殖、EMT以及VM形成,靶向调控基质硬度和YAP可能可以作为胶质瘤治疗的新策略。

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.