基质硬度通过YAP促进胶质瘤细胞上皮-间质转化和血管模拟形成

Q4 Medicine

中华神经外科杂志 Pub Date : 2020-03-28 DOI:10.3760/CMA.J.CN112050-20190307-00081

Jiyang An, Lili Zhou, Zhi-feng Zhang, Xianzhi Liu

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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. \n \n \nResults \nWith 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). 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引用次数: 0

摘要

目的探讨基质硬度促进胶质瘤细胞上皮-间充质转化(EMT)和血管源性模拟(VM)的机制。方法在不同基质硬度条件下培养人胶质瘤细胞株U87。基质刚度0.2、16.0和64.0 kPa分别代表正常脑、胶质瘤和极端刚度。cck法检测U87细胞株的增殖情况。免疫荧光染色检测YAP细胞的定位。实时荧光定量PCR检测YAP靶向基因CTGF、CRY61的表达。Western blot检测YAP的表达和磷酸化表达，以及Akt、ERK1/2等增殖信号。免疫荧光和Western blot检测emt相关蛋白Vimentin、E-cadherin、Twist的表达。在三维培养下，在软硬基质中检测VM形成。用shYAP慢病毒转染U87细胞，检测EMT蛋白的表达和VM的形成。结果随着基质刚度的增加，胶质瘤细胞的增殖活性和Akt、ERK1/2的磷酸化表达增加(均P<0.05)，磷酸化Yap的表达减少(P<0.05)，而CTGF、CRY61的表达水平随着Yap核易位的增加而增加(P<0.05)。免疫荧光染色和Western blot结果显示，随着基质刚度的增加，EMT成分Vimentin和Twist表达水平升高，上皮标志物E-cadherin表达水平降低(均P<0.05)。与软基质3D培养相比，硬基质下U87细胞VM管状结构的形成增多(P<0.05)。干扰Yap表达可抑制胶质瘤细胞增殖增强、Vimentin和Twist表达增强以及基质刚度诱导的VM形成增强(P<0.05)。结论基质刚度通过YAP促进胶质瘤细胞增殖、EMT和VM的形成。靶向基质刚度和YAP可作为胶质瘤治疗的新策略。关键词:胶质瘤;刚度矩阵;Yes-associated蛋白质;Epithelial-mesenchymal过渡;Vasculogenic模仿

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

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

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来源期刊

中华神经外科杂志 Medicine-Surgery

CiteScore

0.10

自引率

0.00%

发文量

10706

期刊介绍： Chinese Journal of Neurosurgery is one of the series of journals organized by the Chinese Medical Association under the supervision of the China Association for Science and Technology. The journal is aimed at neurosurgeons and related researchers, and reports on the leading scientific research results and clinical experience in the field of neurosurgery, as well as the basic theoretical research closely related to neurosurgery.Chinese Journal of Neurosurgery has been included in many famous domestic search organizations, such as China Knowledge Resources Database, China Biomedical Journal Citation Database, Chinese Biomedical Journal Literature Database, China Science Citation Database, China Biomedical Literature Database, China Science and Technology Paper Citation Statistical Analysis Database, and China Science and Technology Journal Full Text Database, Wanfang Data Database of Medical Journals, etc.