NUDT21 alters glioma migration through differential alternative polyadenylation of LAMC1

We identified LAMC1 as a critical NUDT21 target involved in several core glioma-driving signaling pathways. qRT-PCR analysis confirmed that NUDT21-knockdown in GBM cells resulted in the preferred usage of the proximal C/PAS of LAMC1. Furthermore, functional studies revealed that NUDT21-knockdown-induced 3'UTR shortening of LAMC1 was sufficient to induce translational gain, as LAMC1 was upregulated in these cells compared to their respective controls. Additionally, we demonstrated that primary glial cells recapitulated this phenomenon. We also further dissected the miRNA-mediated mechanism by which LAMC1 expression was regulated by showing that NUDT21 knockdown in glioma cells directed the 3'UTR shortening of LAMC1, removing binding sites for miR-124/506 and consequently inducing LAMC1 expression. Remarkably, we reported that the knockdown of NUDT21 significantly promoted GBM cell migration and that co-depletion of LAMC1 and NUDT21 abrogated this effect. Lastly, we observed that LAMC1 3'UTR shortening predicted poor prognosis of low- and high-grade glioma patients from The Cancer Genome Atlas. Overall design: Anti-NUDT21 and scramble shRNA-infected LN229 / U251 human gliablastoma cell lines mRNA 3' end sequencing profiles generated by deep sequencing, in triplicate, using Illumina Nextseq 550.

本研究确认LAMC1是NUDT21的关键靶标，参与多条胶质瘤核心驱动信号通路。定量实时逆转录聚合酶链反应（quantitative real-time reverse transcription PCR, qRT-PCR）分析证实，在胶质母细胞瘤（Glioblastoma, GBM）细胞中敲低NUDT21会使LAMC1的近端切割与多聚腺苷酸化位点（cleavage and polyadenylation site, C/PAS）的使用偏好性升高。功能实验进一步显示，NUDT21敲低诱导的LAMC1的3'非翻译区（3' untranslated region, 3'UTR）缩短足以引发翻译增益，相较于相应对照组细胞，该组细胞中LAMC1的表达水平显著上调。此外，本研究证实原代胶质细胞也重现了这一现象。本研究还进一步解析了miRNA介导的LAMC1表达调控机制：在胶质瘤细胞中敲低NUDT21会导致LAMC1的3'UTR缩短，从而移除miR-124/506的结合位点，最终上调LAMC1的表达。值得注意的是，本研究发现敲低NUDT21可显著促进胶质母细胞瘤细胞的迁移能力，而同时敲低LAMC1与NUDT21则可抵消这一效应。最后，本研究从癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据中观察到，LAMC1的3'UTR缩短可预示低级别与高级别胶质瘤患者的不良预后。实验整体设计：使用Illumina Nextseq 550测序平台，对经抗NUDT21短发卡RNA及阴性对照scrambled shRNA感染的LN229与U251人胶质母细胞瘤细胞系进行mRNA 3'端深度测序，设置三次生物学重复，获取其测序图谱。