BACKGROUND Nuclear 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. METHODS We 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. RESULTS DCX 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. CONCLUSION Collectively, this study highlights a remarkable phenomenon in glioma, hence revealing potential glioma dependencies on DCX expression, which is amenable to targeted therapy. Video abstract.

中文翻译：

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Doublecortin通过RanGTPase信号传导进行核质转运，从而促进神经胶质瘤的进展。

背景技术先前已经报道了几种致癌蛋白的核易位，但是既没有研究双皮质素（DCX）的易位也没有研究涉及的机制。DCX是一种神经元微管相关蛋白（MAP），对成人神经发生和神经元迁移至关重要，并且与神经胶质瘤的不良预后有关。方法我们通过蛋白质印迹法检测了DCX在不同等级的神经胶质瘤组织和常规细胞中的表达。然后，我们分析了Oncomine癌症分析数据库中的表达模式。共聚焦免疫荧光检测细胞隔室中DCX的表达，而亚细胞分离则通过蛋白质印迹法进行检测。脉冲形状高度分析用于验证DCX在较大细胞群中的定位。共免疫沉淀用于检测DCX导入受体的相互作用。为了探测DCX功能，使用CRISPR-Cas9病毒转染生成表达高DCX表达或敲低的稳定细胞，同时使用质粒定点突变体构建体验证通过常规算法预测的假定核定位序列（NLS）并与经典NLS进行比较。进行了计算机内建模，以验证DCX通过选定的假定NLS与输入受体的相互作用。通过Boyden侵袭试验和伤口愈合迁移试验检测DCX高表达，敲低，突变和/或缺失的NLS位点的影响，并通过体外CCK8试验检测存活力，同时在裸鼠中进行异种移植肿瘤模型。结果DCX通过RanGTPase信号转导通路与位于丝氨酸47-酪氨酸70之间N端的NLS进行核质移动。由于神经胶质细胞源性神经营养因子（GDNF）的异常表达，MARK的NLS两侧的丝氨酸47残基的磷酸化可以刺激这种移位。DCX的高表达和核积累可改善体内和体外侵袭性神经胶质瘤的能力。此外，降低或阻断DCX核输入减弱了神经胶质瘤细胞的侵袭性和增殖。结论总体而言，该研究突出了神经胶质瘤中的显着现象，从而揭示了神经胶质瘤对DCX表达的潜在依赖性，这适合于靶向治疗。录像摘要。由于神经胶质细胞源性神经营养因子（GDNF）的异常表达，MARK的NLS两侧的丝氨酸47残基的磷酸化可以刺激这种移位。DCX的高表达和核积累可改善体内和体外侵袭性神经胶质瘤的能力。此外，降低或阻断DCX核输入减弱了神经胶质瘤细胞的侵袭性和增殖。结论总体而言，该研究突出了神经胶质瘤中的显着现象，从而揭示了神经胶质瘤对DCX表达的潜在依赖性，这适合于靶向治疗。录像摘要。由于神经胶质细胞源性神经营养因子（GDNF）的异常表达，MARK的NLS两侧的丝氨酸47残基的磷酸化可以刺激这种移位。DCX的高表达和核积累可改善体内和体外侵袭性神经胶质瘤的能力。此外，降低或阻断DCX核输入减弱了神经胶质瘤细胞的侵袭性和增殖。结论总体而言，该研究突出了神经胶质瘤中的显着现象，从而揭示了神经胶质瘤对DCX表达的潜在依赖性，这适合于靶向治疗。录像摘要。DCX的高表达和核积累可改善体内和体外侵袭性神经胶质瘤的能力。此外，降低或阻断DCX核输入减弱了神经胶质瘤细胞的侵袭性和增殖。结论总体而言，该研究突出了神经胶质瘤中的显着现象，从而揭示了神经胶质瘤对DCX表达的潜在依赖性，这适合于靶向治疗。录像摘要。DCX的高表达和核蓄积改善了体内和体外侵袭性神经胶质瘤的能力。此外，降低或阻断DCX核输入减弱了神经胶质瘤细胞的侵袭性和增殖。结论总体而言，该研究突出了神经胶质瘤中的显着现象，从而揭示了神经胶质瘤对DCX表达的潜在依赖性，这适合于靶向治疗。录像摘要。