Bhargava Shruti, Visvanathan Abhirami, Patil Vikas, Kumar Anuj, Kesari Santosh, Das Saumitra, Hegde Alangar S, Arivazhagan Arimappamagan, Santosh Vani, Somasundaram Kumaravel
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Department of Translational Neuro-Oncology and Neurotherapeutics, Pacific Neuroscience Institute, John Wayne Cancer Institute, Providence Saint John's Health Center, Santa Monica, California, USA.
Oncotarget. 2017 Jun 20;8(25):40469-40485. doi: 10.18632/oncotarget.17118.
The diffusely infiltrative nature of glioblastoma (GBM) makes them highly recurrent. IGF2 mRNA-binding protein 3 (IMP3), a GBM upregulated RNA binding protein, promotes glioma cell migration. An integrative bioinformatics analysis identified p65 (RELA), a subunit of NF-κB heterodimer as a target and an important mediator of IMP3 promoted glioma cell migration. IMP3 increased p65 protein levels without any change in p65 transcript levels, but promoted its polysome association. RIP-PCR demonstrated the binding of IMP3 to p65 transcript. UV crosslinking experiments with in vitro transcribed RNA confirmed the specific and direct binding of IMP3 to sites on p65 3'UTR. Further, IMP3 induced luciferase activity from p65 3'UTR reporter carrying wild type sites but not mutated sites. Exogenous overexpression of p65 from a 3'UTR-less construct rescued the reduced migration of glioma cells in IMP3 silenced condition. In addition, IMP3 silencing inhibited glioma stem-like cell maintenance and migration. The exogenous overexpression of 3'UTR-less p65 significantly alleviated the inhibition of neurosphere formation observed in IMP3 silenced glioma stem-like cells. Further, we show that IMP3 is transcriptionally activated by NF-κB pathway indicating the presence of a positive feedback loop between IMP3 and p65. This study establishes p65 as a novel target of IMP3 in increasing glioma cell migration and underscores the significance of IMP3-p65 feedback loop for therapeutic targeting in GBM.
胶质母细胞瘤（GBM）的弥漫浸润性使其具有高度复发性。IGF2 mRNA结合蛋白3（IMP3）是一种在GBM中上调的RNA结合蛋白，可促进胶质瘤细胞迁移。一项综合生物信息学分析确定，NF-κB异二聚体的一个亚基p65（RELA）是IMP3促进胶质瘤细胞迁移的一个靶点和重要介质。IMP3增加了p65蛋白水平，而p65转录水平没有任何变化，但促进了其多核糖体结合。RIP-PCR证明IMP3与p65转录本结合。用体外转录RNA进行的紫外线交联实验证实了IMP3与p65 3'UTR上的位点具有特异性直接结合。此外，IMP3从携带野生型位点而非突变位点的p65 3'UTR报告基因诱导荧光素酶活性。来自无3'UTR构建体的p65的外源性过表达挽救了IMP3沉默条件下胶质瘤细胞迁移减少的情况。此外，IMP3沉默抑制了胶质瘤干细胞样细胞的维持和迁移。无3'UTR的p65的外源性过表达显著减轻了在IMP3沉默的胶质瘤干细胞样细胞中观察到的神经球形成的抑制。此外，我们表明IMP3被NF-κB途径转录激活，表明IMP3和p65之间存在正反馈环。这项研究确定p65是IMP3在增加胶质瘤细胞迁移中的一个新靶点，并强调了IMP3-p65反馈环在GBM治疗靶向中的重要性。
