MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation

The epithelial-to-mesenchymal (-like) transition (EMT), a crucial embryonic development program, has been linked to the regulation of glioblastoma (GBM) progression and invasion. Here, we investigated the role of <i>MIR517C/miR-517c</i>, which belongs to the <i>C19MC</i> microRNA cluster identified in our preliminary studies, in the pathogenesis of GBM. We found that <i>MIR517C</i> was associated with improved prognosis in patients with GBM. Furthermore, following treatment with the autophagy inducer temozolomide (TMZ) and low glucose (LG), <i>MIR517C</i> degraded <i>KPNA2</i> (karyopherin alpha 2 [RAG cohort 1, importin alpha 1]) and subsequently disturbed the nuclear translocation of TP53 in the GBM cell line U87 in vitro. Interestingly, this microRNA could inhibit autophagy and reduce cell migration and infiltration in U87 cells harboring wild-type (WT) <i>TP53</i>, but not in U251 cells harboring mutant (MU) <i>TP53</i>. Moreover, the expression of epithelial markers (i.e., CDH13/T-cadherin and CLDN1 [claudin 1]) increased, while the expression of mesenchymal markers (i.e., CDH2/N-cadherin, SNAI1/Snail, and VIM [vimentin]) decreased, indicating that the EMT status was blocked by <i>MIR517C</i> in U87 cells. Compared with <i>MIR517C</i> overexpression, <i>MIR517C</i> knockdown promoted infiltration of U87 cells to the surrounding structures in nude mice in vivo. The above phenotypic changes were also observed in <i>TP53</i>^<i>+/+</i> and <i>TP53</i>^{<i>−/−</i>} HCT116 colon cancer cells. In summary, our study provided support for a link between autophagy and EMT status in WT <i>TP53</i> GBM cells and provided evidence for the signaling pathway (<i>MIR517C</i>-KPNA2-cytoplasmic TP53) involved in attenuating autophagy and eliminating the increased migration and invasion during the EMT.

上皮间质（样）转化（Epithelial-to-mesenchymal (-like) transition, EMT）是一类关键的胚胎发育程序，现已被证实与胶质母细胞瘤（glioblastoma, GBM）的进展及侵袭调控密切相关。本研究针对前期筛选得到的、隶属于C19MC微小RNA（microRNA）簇的<i>MIR517C/miR-517c</i>，探讨其在GBM发病机制中的作用。研究发现，<i>MIR517C</i>的表达与GBM患者的良好预后相关。进一步实验显示，经自噬诱导剂替莫唑胺（temozolomide, TMZ）与低糖（low glucose, LG）处理后，<i>MIR517C</i>可降解核转运蛋白α2（karyopherin alpha 2 [RAG队列1, 输入蛋白α1]，KPNA2），并在体外实验中干扰U87胶质瘤细胞内TP53的核转位。有趣的是，该微小RNA可在携带野生型（wild-type, WT）TP53的U87细胞中抑制自噬、降低细胞迁移与浸润能力，但在携带突变型（mutant, MU）TP53的U251细胞中无此效应。此外，上皮标志物（即CDH13/T-钙粘蛋白与CLDN1/紧密连接蛋白1）的表达水平上调，而间质标志物（即CDH2/N-钙粘蛋白、SNAI1/Snail与VIM/波形蛋白）的表达水平下调，表明<i>MIR517C</i>可阻断U87细胞的EMT进程。与<i>MIR517C</i>过表达相比，<i>MIR517C</i>敲低可在体内裸鼠实验中促进U87细胞向周围结构浸润。上述表型变化同样在TP53^<i>+/+</i>与TP53^{<i>−/−</i>}的HCT116结肠癌细胞中得以验证。综上，本研究证实了野生型TP53的GBM细胞中自噬与EMT状态之间的关联，并为<i>MIR517C</i>-KPNA2-胞质TP53信号通路在减弱自噬、消除EMT过程中增强的迁移与侵袭能力提供了实验依据。