Department of Neurobiology, School of Life Sciences, China Medical University, Shenyang, 110122, China.
Liaoning University of Traditional Chinese Medicine, Shenyang, 110034, China.
Cell Biol Toxicol. 2024 Jun 11;40(1):44. doi: 10.1007/s10565-024-09890-5.
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.
The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM.
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.
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.
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.
血管生成拟态（VM）是一种神秘的生理特征，影响胶质母细胞瘤（GBM）肿瘤内的血液供应，从而维持其生长。先前的研究表明 NFATC3、FOSL1 和 HNRNPA2B1 是血管生成中关键因子 VEGFR2 的重要介质，它们的分子关系对于 GBM 中的 VM 可能至关重要。
本研究旨在了解 NFATC3、FOSL1 和 HNRNPA2B1 如何共同影响 GBM 中的 VM。
我们在体外和体内研究了 U251 和 U373 胶质母细胞瘤细胞系中 VM 的潜在基因调控机制。体外细胞基础实验研究了 NFATC3、FOSL1 和 HNRNPA2B1 在 RNA 干扰（RNAi）介导的敲低以及相应对照情况下，对 GBM 细胞增殖、VM 和迁移的作用。Western blot 和 qRT-PCR 实验检测了 VEGFR2 的表达水平。CO-IP 用于检测蛋白-蛋白相互作用，ChIP 用于检测 DNA-蛋白复合物，RIP 用于检测 RNA-蛋白复合物。组织化学染色用于检测体内 VM 管形成。
聚焦于 NFATC3、FOSL1 和 HNRNPA2B1，我们发现它们在 GBM 中均显著上调，并与 U251 和 U373 细胞系中类似 VM 的细胞行为呈正相关。NFATC3、FOSL1 或 HNRNPA2B1 的敲低均导致关键生长因子基因 VEGFR2 水平降低，从而抑制增殖、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实验表明，NFATC3 结合 VEGFR2 启动子区域以增强 VEGFR2 基因表达。值得注意的是，FOSL1 作为共因子与 NFATC3 相互作用，增强 NFATC3 的 DNA 结合能力，从而增强类似 VM 的细胞行为。此外，通过 HNRNPA2B1 结合 NFATC3 mRNA，细胞内 NFATC3 蛋白水平得到增强。此外，在 GBM 细胞中通过 RNAi 沉默 NFATC3、FOSL1 和 HNRNPA2B1 降低了它们在体内形成肿瘤和类似 VM 行为的能力。
综上所述，我们的研究结果表明，NFATC3 通过与 HNRNPA2B1 和 FOSL1 的分子和上位相互作用，成为 GBM 肿瘤生长的重要介质，从而影响 VEGFR2 的表达和类似 VM 的细胞行为。
