PDF(Pubmed)
Abstract：
BACKGROUND: Vasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM.
OBJECTIVE: The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM.
METHODS: We have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein-protein interactions, ChIP was used to detect DNA-protein complexes, and RIP was used to detect RNA-protein complexes. Histochemical staining was used to detect VM tube formation in vivo.
RESULTS: Focusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo.
CONCLUSIONS: Taken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.
OBJECTIVE: The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM.
METHODS: We have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein-protein interactions, ChIP was used to detect DNA-protein complexes, and RIP was used to detect RNA-protein complexes. Histochemical staining was used to detect VM tube formation in vivo.
RESULTS: Focusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo.
CONCLUSIONS: Taken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.
摘要：
背景：血管生成拟态（VM）是一种神秘的生理特征，它影响胶质母细胞瘤（GBM）肿瘤的持续生长的血液供应。以前的研究确定NFATC3，FOSL1和HNRNPA2B1是VEGFR2的重要介导因子，它们的分子关系可能对GBM中的VM至关重要。
目的：本研究的目的是了解NFATC3，FOSL1和HNRNPA2B1如何共同影响GBM中的VM。
方法：我们在体外和体内研究了GBM细胞系U251和U373中VM的潜在基因调控机制。进行体外基于细胞的测定以探索NFATC3，FOSL1和HNRNPA2B1在GBM细胞增殖中的作用，VM和迁移，在RNA干扰（RNAi）介导的敲低以及相应的对照的背景下。使用蛋白质印迹和qRT-PCR测定来检查VEGFR2表达水平。CO-IP用于检测蛋白质-蛋白质相互作用，ChIP用于检测DNA-蛋白质复合物，RIP用于检测RNA-蛋白质复合物。组织化学染色用于检测体内VM管的形成。
结果：关注NFATC3，FOSL1和HNRNPA2B1，我们发现它们在GBM中都显着上调，并且与U251和U373细胞系中的VM样细胞行为呈正相关。NFATC3，FOSL1或HNRNPA2B1的敲除均导致VEGFR2水平降低，VEGFR2是驱动VM的关键生长因子基因，以及抑制增殖，细胞迁移和体外VM活性。染色质免疫沉淀（ChIP）研究和荧光素酶报告基因测定显示，NFATC3与VEGFR2的启动子区结合以增强VEGFR2基因表达。值得注意的是,FOSL1作为辅因子与NFATC3相互作用，以增强NFATC3的DNA结合能力，从而增强VM样细胞行为。此外，细胞中NFATC3蛋白的水平通过HNRNPA2B1与NFATC3mRNA的结合而增强。此外，RNAi介导的GBM细胞中NFATC3，FOSL1和HNRNPA2B1的沉默降低了其体内肿瘤形成和VM样行为的能力。
结论：综合来看，我们的研究发现NFATC3通过其与HNRNPA2B1和FOSL1的分子和上位性相互作用来影响VEGFR2的表达和VM样细胞行为,从而确定NFATC3是GBM肿瘤生长的重要介质。
目的：本研究的目的是了解NFATC3，FOSL1和HNRNPA2B1如何共同影响GBM中的VM。
方法：我们在体外和体内研究了GBM细胞系U251和U373中VM的潜在基因调控机制。进行体外基于细胞的测定以探索NFATC3，FOSL1和HNRNPA2B1在GBM细胞增殖中的作用，VM和迁移，在RNA干扰（RNAi）介导的敲低以及相应的对照的背景下。使用蛋白质印迹和qRT-PCR测定来检查VEGFR2表达水平。CO-IP用于检测蛋白质-蛋白质相互作用，ChIP用于检测DNA-蛋白质复合物，RIP用于检测RNA-蛋白质复合物。组织化学染色用于检测体内VM管的形成。
结果：关注NFATC3，FOSL1和HNRNPA2B1，我们发现它们在GBM中都显着上调，并且与U251和U373细胞系中的VM样细胞行为呈正相关。NFATC3，FOSL1或HNRNPA2B1的敲除均导致VEGFR2水平降低，VEGFR2是驱动VM的关键生长因子基因，以及抑制增殖，细胞迁移和体外VM活性。染色质免疫沉淀（ChIP）研究和荧光素酶报告基因测定显示，NFATC3与VEGFR2的启动子区结合以增强VEGFR2基因表达。值得注意的是,FOSL1作为辅因子与NFATC3相互作用，以增强NFATC3的DNA结合能力，从而增强VM样细胞行为。此外，细胞中NFATC3蛋白的水平通过HNRNPA2B1与NFATC3mRNA的结合而增强。此外，RNAi介导的GBM细胞中NFATC3，FOSL1和HNRNPA2B1的沉默降低了其体内肿瘤形成和VM样行为的能力。
结论：综合来看，我们的研究发现NFATC3通过其与HNRNPA2B1和FOSL1的分子和上位性相互作用来影响VEGFR2的表达和VM样细胞行为,从而确定NFATC3是GBM肿瘤生长的重要介质。
