ZDHHC12在胶质母细胞瘤中通过YAP1调控肿瘤特性

doi:10.19401/j.cnki.1007-3639.2022.06.007

摘要/Abstract

摘要：

背景和目的：胶质母细胞瘤（glioblastoma,GBM）是目前常见的恶性脑部肿瘤之一,但GBM相关发病机制仍不完全清楚。本研究旨在分析锌指DHHC结构域蛋白（zinc finger DHHC domain-containing protein,ZDHHC）12对Yes相关蛋白1（Yes-associated protein 1,YAP1）的调控,以及ZDHHC12/YAP1轴对GBM肿瘤特性的调控。方法：在癌症基因组图谱（The Cancer Genome Atlas,TCGA）数据库以及基因型-组织表达（Genotype-Tissue Expression,GTEx）数据库中分析ZDHHC12分别在正常脑组织及GBM中的表达情况,运用蛋白质印迹法（Western blot）以及实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测,分析在U87、U251以及人正常星形胶质细胞（NHA）的mRNA表达及蛋白水平。在U87和U251两种GBM细胞系中通过设计的小干扰RNA（small interfering RNA,siRNA）敲低ZDHHC12,并验证YAP1蛋白水平的变化。通过免疫共沉淀（co-immunoprecipitation,Co-IP）验证内源性及外源性ZDHHC12及YAP1的相互关系。通过细胞计数试剂盒-8（cell counting kit-8,CCK-8）实验、细胞平板克隆形成实验、划痕实验分析GBM细胞敲低ZDHHC12后在增殖及迁移能力上发生的改变。检测在敲低ZDHHC12后GBM细胞系中上皮-间质转化（epithelial-mesenchymal transition,EMT）标志物变化。分析ZDHHC12对GBM患者预后的影响。并检测在不同组织样本中ZDHHC12和YAP1蛋白水平。结果：TCGA数据库及GTEx数据库分析结果显示,ZDHHC12在GBM中的表达量明显高于正常脑组织（P<0.01）,GBM细胞系中ZDHHC12的mRNA表达及蛋白水平高于NHA细胞系。U87和U251两组GBM细胞系中ZDHHC12的敲低会引起YAP1蛋白水平的降低。Co-IP实验验证了ZDHHC12及YAP1蛋白的相互关系。敲低ZDHHC12的表达能够显著抑制GBM细胞的增殖和迁移能力,差异有统计学意义（P<0.05）。ZDHHC12的敲低也可以引起EMT相关标志物的改变。YAP1的回复可以逆转敲低ZDHHC12所引起的GBM肿瘤特性变化。TCGA数据库中,ZDHHC12的表达也与GBM患者的预后密切相关。在组织中ZDHHC12的表达也与YAP1表达呈高度正相关。结论：ZDHHC12在GBM中高表达且与YAP1呈正相关,ZDHHC12/YAP1轴可以调控GBM相关肿瘤特性。

关键词: 胶质母细胞瘤, 胶质母细胞瘤, 锌指DHHC结构域蛋白12, Yes相关蛋白1, 上皮-间质转化

Abstract:

Background and purpose: Glioblastoma (GBM) is one of the most malignant brain tumors, however, the pathogenesis of GBM has not been thoroughly studied. The purpose of this study was to analyze the regulation of Yes-associated protein 1 (YAP1) by zinc finger DHHC domain-containing protein 12 (ZDHHC12), and the regulation of GBM tumor characteristics through the ZDHHC12/YAP1 axis. Methods: The expression of ZDHHC12 in normal brain tissue and GBM was analyzed in the The Cancer Genome Atlas (TCGA) database and Genotype-Tissue Expression (GTEx) database. Western blot and real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) were used to detect the mRNA expression and protein levels in U87, U251 and NHA. ZDHHC12 was knocked down by designed siRNA in two GBM cell lines, U87 and U251, and the protein levels of YAP1 were detected. The interaction between endogenous and exogenous ZDHHC12 and YAP1 was verified by co-immunoprecipitation (Co-IP). The changes in proliferation and migration ability of GBM cells after knockdown of ZDHHC12 and restoration of YAP1 were analyzed by cell counting kit-8 (CCK-8) assay, cell plate clone formation assay and scratch assay. Changes in markers of epithelial-mesenchymal transition (EMT) in GBM cell lines after knockdown of ZDHHC12 and restoration of YAP1 were detected. The effect of ZDHHC12 on the prognosis of GBM patients was analyzed, and detection of the protein levels of ZDHHC12 and YAP1 in different tissue samples was carried out. Results: The analysis results of TCGA database and GTEx database showed that the expression of ZDHHC12 was significantly higher in GBM than in normal brain tissue (P<0.01), and the mRNA and protein levels of ZDHHC12 were higher in GBM cell lines than in NHAs cell lines. Knockdown of ZDHHC12 in both U87 and U251 GBM cell lines caused a decrease in YAP1 protein levels. Co-IP experiments verified the interaction between ZDHHC12 and YAP1 proteins. Knockdown of ZDHHC12 could significantly inhibit the proliferation and migration of GBM cells, and the difference was statistically significant (P<0.05). Knockdown of ZDHHC12 also caused changes in EMT-related markers. Restoration of YAP1 reversed the changes in GBM tumor properties induced by the knockdown of ZDHHC12. In the TCGA database, the expression of ZDHHC12 was also closely related to the prognosis of GBM patients. The expression of ZDHHC12 in tissues was positively correlated with YAP1 expression. Conclusion: ZDHHC12 is highly expressed in GBM and positively related to YAP1, and the ZDHHC12/YAP1 axis regulates GBM tumor characteristics.

Key words: Glioblastoma, Zinc finger DHHC domain-containing protein 12, Yes-associated protein 1, Epithelial-mesenchymal transition

中图分类号:

R739.4

张起翔, 尤永平. ZDHHC12在胶质母细胞瘤中通过YAP1调控肿瘤特性[J]. 中国癌症杂志, 2022, 32(6): 527-534.

ZHANG Qixiang, YOU Yongping. ZDHHC12 regulates tumor properties through YAP1 in glioblastoma[J]. China Oncology, 2022, 32(6): 527-534.

图/表 4

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