YB-1选择性清除miR-125b/外泌体促进肿瘤干细胞异质性演化：脑胶质瘤放疗抵抗的新伎俩？

Proneural (PN) and mesenchymal (MES) glioma stem cells represent two mutally-exclusive, and biologically distinct GSC subtype. Intriguingly, experimental and clinical data suggest that upon radiation treatment, PN GSCs gain the phenotypes of MES GSCs, which are much more aggressive and radioresistant than original PN GSCs. Huang et al. report that miR-125b inhibits mesenchymal differentiation and therefore prevents glioblastoma radioresistance by a regulatory circuit FZD6/wnt. Radiation treatment decreases the intracellular expression of miR-125b, leading to induced MES markers and reduced PN gene. However, the exact mechanisms remain poorly understood. Our latest exciting results demonstrate that radiation induced the release of miR-125b enriched exosome from GSCs. In addition, Y-box binding protein 1 (YB-1), the principal protein that binds to RNA, is upregulated in irradiated PN GSCs. Inhibition of YB-1 effectively abrogates the proneural-mesenchymal transformation during radiation. Based on these findings, we hypothesize that YB-1 eliminates miR-125b through the release of cargo-bearing exosomes and drives proneural GCSs shift towards a more aggressive phenotype, which is responsible for recurrent GBMs and their resistance to radiation therapy. Thus, the overall objective of this grant proposal is to determine the mechanisms by which the upregulation of YB-1 facilitates disease progression. We will also validate these mechanisms in clinical outcomes of patients undergoing radiotherapy treatment for glioblastoma. In Aim 1, we will determine the critical role of YB-1 in package of miR-125b enriched exosomes and purging intracellular miRNA during irradiation. Aim 2 will identify the release of miR-125b enriched exosomes and its effect on phenotypes of post-therapeutic glioblastoma tumors as well as DNA damage repair and tumor radiosensitivity. The focus of Aim 3 will be to seek the correlation of serum miR-125b positive exosomes and YB-1 expression in tumor tissue with the prognosis of glioblastoma patients. These studies will greatly enhance our knowledge of the molecular mechanism that YB-1 drives proneural-mesenchymal transformation of glioma stem cells, which is pivotal for the development of new therapeutic strategies to prevent radioresistance and subsequently glioblastoma recurrence.

前神经元-间质转化（PMT）是胶质瘤干细胞（GSCs）异质演变的重要形式，与辐射抵抗和肿瘤复发密切相关。我们发现：体外诱导PMT过程中GSCs胞内YB-1高表达，且伴随着miR-125b/外泌体的大量释放；靶向抑制YB-1能有效阻断PMT，提高GSCs放射敏感性。据此我们推测：作为核酸类物质的重要结合蛋白，YB-1可能通过外泌体选择性清除miR-125b，打破维持GSCs前神经元表型的FZD6/Wnt正反馈环，促进其间质转化，最终导致放疗抵抗。本项目拟原代培养GSCs，应用分子克隆和基因编辑技术解析YB-1的表达及其磷酸化水平对miR-125b/外泌体的组装与清除、Wnt信号、DNA损伤修复及放射敏感性的影响，厘清血清miR-125b/外泌体与GBM患者放疗疗效之间的联系，阐明YB-1选择性清除miR-125b/外泌体、促进PMT、诱导放射抵抗的分子机制，进而为GBM辐射增敏提供新靶点。

异质性是恶性胶质瘤复杂难治的关键原因，异质性演化则是肿瘤细胞与环境胁迫因素博弈的结果，是肿瘤细胞获得治疗抵抗的重要途径。对于 GBM 而言，前神经元型胶质瘤干细胞 （PN GSCs）在治疗压力下异质性演化成恶性程度更高的间质型（MES），抵抗常规放疗，这意味着靶向阻断 PMT 有望提高胶质瘤患者的辐射敏感性，防止复发。本项目以“体外诱导 PMT过程中 YB-1 促进 GSCs 释放 miR-125b/外泌体”这一现象为切入点，分别从动物、细胞以及分子水平全面的阐明了电离辐射刺激巨噬细胞进入GBM病灶及周围，通过在肿瘤局部释放TNFα活化GSCs内NF-κB信号通路，进而促进GSCs PMT演化进程，最终诱导GBM肿瘤放疗抵抗这一全新的机制。基于上述发现，进一步利用细胞涂层纳米技术创新性的构建基于巨噬细胞膜的负载NBD多肽仿生纳米颗粒，靶向干预 YB-1及巨噬细胞。在体内外实验均证实该纳米颗粒能够释放NBD多肽靶向阻遏GSCs内NF-κB信号通路的激活，进而阻断GSCs PMT演化进程，最终实现GBM辐射增敏的效应。本研究为抑制放疗后GBM的放疗抵抗表型以及防治肿瘤复发提供了新的方向。