RAN directly binds and stabilizes G3BP1 mRNA in the nucleus to facilitate proliferation and metastasis of nasopharyngeal carcinoma (anti-RAN RIP-seq)

RNA binding proteins (RBPs) play a critical role in tumor progression by participating in the post-transcriptional regulation of RNA. However, the expression and function of RBPs in nasopharyngeal carcinoma (NPC) remain elusive. This study identified 5 RBPs (RAN, EZH2, RDM1, HRSP12, and ALYREF) that were up-regulated in NPC and could promote NPC cells migration or proliferation. RAN was first investigated because of its most significant effect on NPC cells. Functionally, RAN facilitated NPC proliferation, migration, and invasion in vitro and in vivo. High expression of RAN was associated with poor prognosis of NPC patients and could be performed as a prognostic biomarker. Mechanistically, RAN mediated the nucleus import of TDP43 and enhanced TDP43 nuclear distribution. On the other hand, RAN was directly bound to the coding sequence of G3BP1 mRNA and served as an adapter to facilitate TDP43 interacting with G3BP1 mRNA 3’untranslated region. Thereby increased G3BP1 mRNA stability in the nucleus and led to up-regulation of G3BP1, which further enhanced ATK/ERK phosphorylation and ultimately promoted NPC proliferation and metastasis. RIP-seq was performed to identify downstream RNAs interacting with RAN in two NPC cell lines HONE-1 and SUNE-1 respectively. Cell lysates were incubated with protein A/G magnetic beads coated with 5 μg of RAN antibody overnight at 4 °C. The immunoprecipitated RNA was separated with elution buffer and purified for next-generation sequencing.

RNA结合蛋白（RNA binding proteins, RBPs）通过参与RNA的转录后调控，在肿瘤进展中发挥关键作用。然而，RNA结合蛋白在鼻咽癌（nasopharyngeal carcinoma, NPC）中的表达与功能仍有待阐明。本研究鉴定出5种在鼻咽癌中呈上调表达的RNA结合蛋白（RAN、EZH2、RDM1、HRSP12及ALYREF），它们可促进鼻咽癌细胞的迁移与增殖。其中，RAN对鼻咽癌细胞的调控效应最为显著，因此率先对其展开研究。功能实验证实，RAN可在体内外促进鼻咽癌细胞的增殖、迁移与侵袭。RAN高表达与鼻咽癌患者的不良预后密切相关，可作为预后生物标志物。从机制层面来看，RAN可介导TDP43的核输入，增强TDP43的核分布。另一方面，RAN可直接结合G3BP1 mRNA的编码序列，并作为适配器促进TDP43与G3BP1 mRNA的3'非翻译区（3' untranslated region, 3'UTR）相互作用，从而提升G3BP1 mRNA在细胞核内的稳定性，上调G3BP1的表达水平，进而增强AKT/ERK磷酸化，最终促进鼻咽癌的增殖与转移。本研究分别在两种鼻咽癌细胞系HONE-1与SUNE-1中开展RNA免疫沉淀测序（RNA immunoprecipitation sequencing, RIP-seq），以鉴定与RAN结合的下游RNA。具体操作步骤为：将细胞裂解液与包被有5 μg RAN抗体的蛋白A/G磁珠在4℃条件下孵育过夜；使用洗脱缓冲液分离免疫沉淀得到的RNA，纯化后进行下一代测序（next-generation sequencing, NGS）。