长链非编码RNA EF565083在胶质瘤干细胞"干性"产生和维持中的作用机制研究

Gliomas are the most common primary brain tumors in adults. Despite new biological insights and advances in therapy, the prognosis of patients with gliomas still remains poor in the last decades. Cancer stem cell hypothesis posits that a subpopulation of cancer cells is highly enriched with tumorigenic potential. Compared with bulk tumor cells, glioma stem cells (GSCs) survive better against irradiation and chemotherapies, thereby contributing to therapeutic resistance and tumor recurrence. The "stemness" is required for maintenance of multipotency and survival in GSCs, and plays an important role in the occurrence and development of gliomas. Accumulating evidence recently suggests that long non-coding RNAs (lncRNAs) have essential roles in tumorigenesis, and that lncRNA-mediated biology occupies a central place in cancer progression. Our previous study demonstrated that expression of lncRNA-EF565083 was significantly down-regulated in the process of GSC differentiation, and over-expression of lncRNA-EF565083 can recover some stem cell properties in differentiated glioma cells. Accordingly, we hypothesize that lncRNA-EF565083 plays an important role in the induction and maintenance of GSC "stemness" by regulating Sox2 and PRC2. This subject specifically aims to: 1. uncover the role of lncRNA-EF565083 in induction of "stemness" in differentiated glioma and glial cells. 2. discover the function of lncRNA-EF565083 for maintaining "stemness" of GSCs, and investigate whether modulation of lncRNA-EF565083 expression could serve as a potential therapeutic strategy in glioma; 3. reveal the mechanism that lncRNA-EF565083 regulate GSCs by targeting Sox2 and PRC2; 4. detect whether lncRNA-EF565083 could serve as a potential clinical prognostic markers in glioma. This subject will explore the potential mechanisms underlying the generation and maintenance of the GSC "stemness", promote the understandings of molecular mechanisms of gliomas, and provide new avenues in the development of effective therapeutics against GSCs to improve gliomas treatment.

胶质瘤干细胞（GSC）在胶质瘤发生发展中发挥着重要作用。我们前期研究发现长链非编码RNA（lncRNA）EF565083的表达在GSC分化过程中显著变化，上调EF565083可使分化的胶质瘤细胞重新获得部分干细胞性能；据此，我们设想EF565083可能通过调控Sox2和PRC2对GSC"干性"的产生和维持发挥着重要作用。故本课题拟：1.研究EF565083诱导胶质瘤细胞和胶质细胞去分化产生"干性"的作用，探讨GSC发生的可能机制；2.研究EF565083维持GSC"干性"的作用，探讨EF565083是否可以作为潜在的胶质瘤治疗靶点；3.揭示EF565083通过调控Sox2和PRC2对GSC发挥作用的机制；4.评估EF565083是否可以作为潜在的胶质瘤预后标记物。该课题从lncRNA角度探索GSC"干性"产生和维持的作用机制，对开发新的胶质瘤治疗方法具有一定的意义。

胶质瘤是中枢神经系统最常见的恶性肿瘤，尽管治疗手段不断改进，但收效甚微，临床对其缺乏有效的治疗手段。胶质瘤干细胞不仅很好地解释了胶质瘤的起源问题，而且也说明了常规治疗方法无法取得明显疗效的原因，对正确认识和理解胶质瘤的发生发展机制有着十分重要的理论意义和实际应用价值。本研究成功分离鉴定出胶质瘤干细胞（GSCs）和胶质瘤细胞。分别在体内外感染慢病毒载体上调或抑制lncRNA-EF565083的表达，检测胶质瘤细胞和胶质细胞去分化和获得自我更新的变化、裸鼠颅内成瘤能力差异，评估lncRNA-EF565083 作为GSCs 治疗靶点的潜能。检测lncRNA-EF565083 与EZH2和LSD1的结合调控关系，并联合应用EZH2和LSD1 的特异性siRNA，确定EZH2和LSD1在lncRNA-EF565083 影响GSCs“干性”通路中的必要作用。通过检测本单位保存的大样本胶质瘤标本中lncRNA-EF565083 的表达情况，分析lncRNA-EF565083 与胶质瘤病理级别和患者预后等指标的关联性，明确了lncRNA-EF565083作为潜在的临床胶质瘤预后指示标记物的价值。在此基础上，首次利用生物信息学分析lncRNA 和mRNA 在GSCs 和分化胶质瘤细胞中的差异表达谱，锁定了12 个lncRNAs，逐步排除转染确定lncRNA-EF565083、LINC00277 和XLOC-012683是转化过程所必须的。通过RIP 实验证明LINC00277 存在与MLL1 的结合关系，并且过表达三种lncRNA的混合体可显著降低胶质瘤细胞中的H3K27me3 水平。此外，首次在临床大样本中验证miR-196a在胶质母细胞瘤组织中表达水平显著上升，验证miR-196a是胶质母细胞瘤患者预后的独立预测指标，明确miR-196a抑制剂可有效治疗异体胶质瘤移植裸鼠肿瘤生长。首次研究证明miR-300在胶质瘤组织和胶质瘤干细胞中的表达水平显著上升，并且miR-300可显著促进胶质瘤干样细胞自我更新和抑制分化。明确了EGF +61G/A多态性和胶质瘤易感性存在显著相关性，并与胶质瘤恶性进展显著相关。总结临床一例同时表现良性和恶性肿瘤特征的多发脑膜瘤，并验证其发生可能源自肿瘤干细胞。以上研究结果对探索GSCs发生维持的机制，发现新的胶质瘤治疗靶点具有积极的意义。