Doublecortin undergo nucleocytoplasmic transport via the RanGTPase signaling to promote glioma progression
Doublecortin undergo nucleocytoplasmic transport via the RanGTPase signaling to promote glioma progression
复制标题
双皮质素通过 RanGTPase 信号传导进行核细胞质转运，促进神经胶质瘤进展
DOI：
10.1186/s12964-019-0485-5
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发表时间：
2020-02-12
影响因子：
8.4
通讯作者：
Gao, Dianshuai
中科院分区：
文献类型：
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作者：
Ayanlaja, Abiola Abdulrahman;Ji, Guanquan;Gao, Dianshuai
背景以往已有报道几种致癌蛋白质的核转位，但双皮质素（doublecortin，DCX）的核转位及其机制尚未见报道。DCX是一种神经元微管相关蛋白（MAP），这是至关重要的成人神经发生和神经元迁移，并已与预后不良的胶质瘤。MethodsWe探测DCX表达在不同级别的胶质瘤组织和常规细胞通过Western印迹。然后我们分析了Oncomine癌症谱数据库中的表达模式。共聚焦免疫荧光法用于检测细胞区室中的DCX表达，而亚细胞分级分离通过蛋白质印迹法进行探测。脉冲形状高度分析用于验证DCX在更大细胞群中的定位。免疫共沉淀用于检测DCX-输入受体相互作用。为了探测DCX功能，使用CRISPR-Cas9病毒转染产生表达高DCX表达或敲低的稳定细胞，而质粒定点突变构建体用于验证通过常规算法预测的推定核定位序列（NLS）并与经典NLS进行比较。进行计算机模拟以验证DCX与输入受体通过所选推定NLS的相互作用。DCX的高表达，敲低，突变，和/或删除推定的NLS网站的影响进行了探测通过Boyden的侵袭试验和伤口愈合迁移试验，并通过CCK 8检测在体外的活力，而异种移植瘤模型进行了裸鼠。ResultsDCX通过RanGTclase信号通路与NLS位于N-末端之间的丝氨酸47-酪氨酸70经历核质运动。这种易位可以刺激MARK的丝氨酸47残基侧翼的NLS由于异常表达的胶质细胞源性神经营养因子（GDNF）的磷酸化。DCX的高表达和核积聚提高了胶质瘤在体外和体内的侵袭能力。此外，敲低或阻断DCX核输入减弱胶质瘤细胞的侵袭性和增殖。ConclusionCollectively，这项研究突出了胶质瘤中的一个显着现象，从而揭示了胶质瘤对DCX表达的潜在依赖性，这是适合靶向治疗的。
BackgroundNuclear translocation of several oncogenic proteins have previously been reported, but neither the translocation of doublecortin (DCX) nor the mechanism involved has been studied. DCX is a neuronal microtubule-associated protein (MAP) that is crucial for adult neurogenesis and neuronal migration and has been associated with poor prognosis in gliomas.MethodsWe probed DCX expression in different grades of glioma tissues and conventional cells via western blotting. Then we analyzed the expression pattern in the Oncomine cancer profiling database. Confocal Immunofluorescence was used to detect DCX expression in the cellular compartments, while subcellular fractionation was probed via western blotting. Pulse shape height analysis was utilized to verify DCX localization in a larger population of cells. Co-immunoprecipitation was used in detecting DCX-import receptors interactions. To probe for DCX functions, stable cells expressing high DCX expression or knockdown were generated using CRISPR-Cas9 viral transfection, while plasmid site-directed mutant constructs were used to validate putative nuclear localization sequence (NLS) predicted via conventional algorithms and comparison with classical NLSs. in-silico modeling was performed to validate DCX interactions with import receptors via the selected putative NLS. Effects of DCX high expression, knockdown, mutation, and/or deletion of putative NLS sites were probed via Boyden's invasion assay and wound healing migration assays, and viability was detected by CCK8 assays in-vitro, while xenograft tumor model was performed in nude mice.ResultsDCX undergoes nucleocytoplasmic movement via the RanGTPase signaling pathway with an NLS located on the N-terminus between serine47-tyrosine70. This translocation could be stimulated by MARK's phosphorylation of the serine 47 residue flanking the NLS due to aberrant expression of glial cell line-derived neurotrophic factor (GDNF). High expression and nuclear accumulation of DCX improve invasive glioma abilities in-vitro and in-vivo. Moreover, knocking down or blocking DCX nuclear import attenuates invasiveness and proliferation of glioma cells.ConclusionCollectively, this study highlights a remarkable phenomenon in glioma, hence revealing potential glioma dependencies on DCX expression, which is amenable to targeted therapy.Video abstract Graphical Abstract