Systematic dissection of function and mechanism of long non-coding RNAs in glioblastoma

项目总结/摘要 胶质母细胞瘤（GBM）是一种高级别胶质瘤（IV级），是最常见和最恶性的原发性脑肿瘤 在成年人中。GBM没有有效的治疗方法。标准治疗后，GBM的中位生存期 患者约15个月。GBM的特征在于增强的细胞增殖，高的细胞增殖倾向， 侵袭/弥漫性浸润整个脑，以及对化疗/放疗的抗性。新兴 证据支持长的非编码RNA（lncRNA，约18，000个人类lncRNA基因）可以介导肿瘤- 促进/抑制作用，并作为独立的诊断/预后生物标志物。我们以前的 整合基因组研究显示，许多lncRNA表现出表达失调， 临床预后，或在GBM中频繁发生体细胞拷贝数改变，表明 lncRNA在GBM发病机制中的作用然而，大多数lncRNA在GBM中的功能和机制尚不清楚。 为了填补这一空白，我们的长期目标是利用系统的多组学方法来表征功能 和GBM中lncRNA的机制，并帮助开发基于新见解的新治疗策略 GBM中lncRNA的调节。我们使用CRISPR干扰（CRISPRi）进行的大规模功能丧失筛查 鉴定了一种对GBM细胞生长至关重要的反义lncRNA，lnc-YINC（YBX 1相互作用lncRNA）， 与正常脑组织和低级别胶质瘤（LGG）相比，GBM中的表达显著上调。的 lnc-YINC的高表达与GBM患者的总生存期较短相关。从功能上讲，lnc- YINC是GBM细胞/胶质瘤干细胞（GSC）细胞周期进程的关键调控因子 和GSC的自我更新。它还保护GBM细胞/GSC免受DNA双链断裂（DSB）引起的损伤。 由内源性压力或外源性电离辐射引起。lnc-YINC与YBX 1相互作用的机制， 转录后调节细胞周期或DSB修复的关键调节因子的表达，至少部分通过 稳定它们的mRNA基于这些新的发现，我们假设lnc-YINC是促进GBM的 通过增强YBX 1促进GBM细胞/GSC增殖并调节其放射敏感性的lncRNA 与细胞周期/DSB修复的关键调节因子结合，并在转录后水平共调节其表达。 本研究的目的是：（a）确定lnc-YINC在胶质瘤发生中的作用，（B）整合 增强型CLIP-seq（eCLIP-seq）、核糖体分析、RNA-seq和定量质谱数据， 功能性试验，以确定lnc-YINC/YBX 1的下游靶点，这些靶点是调节细胞周期的关键， DSB修复，（c）剖析lnc-YINC/YBX 1轴转录后的分子机制， 调节细胞周期或DSB修复的关键调节因子的表达，（d）确定lnc-YINC在 调节GBM细胞/GSC的放射敏感性，并研究联合 用放射疗法抑制lnc-YINC对体内GBM维持。该应用程序通过以下方式得到加强： GBM基础/临床科学、DNA损伤修复、RNA生物学和蛋白质组学专家团队。

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM), a high-grade glioma (grade IV), is the most prevalent and malignant primary brain tumor in adults. There is no effective treatment of GBM. After standard treatment, the median survival of GBM patients is around 15 months. GBM is featured by enhanced cell proliferation, high propensity of invasion/diffuse infiltration throughout the brain, and resistance to chemo-/radiation therapy. Emerging evidence supports that long non-coding RNAs (lncRNAs, ~18,000 human lncRNA genes) can mediate tumor- promoting/suppressing effects and serve as independent diagnostic/prognostic biomarkers. Our previous integrative genomic study revealed that many lncRNAs show dysregulated expression, are associated with clinical prognosis, or harbor frequent somatic copy number alterations in GBM, suggesting an important role of lncRNAs in GBM pathogenesis. However, the function and mechanism of most lncRNAs in GBM are unknown. To fill this gap, our long-term goal is to leverage systematic multi-omic approaches to characterize the function and mechanism of lncRNAs in GBM, and to help develop new therapeutic strategies based on novel insight into lncRNA regulation in GBM. Our large-scale loss-of-function screen using CRISPR interference (CRISPRi) identified an antisense lncRNA, lnc-YINC (YBX1-interacting lncRNA) that was critical for GBM cell growth and was significantly up-regulated in GBM compared with normal brain tissue and low-grade glioma (LGG). The higher expression of lnc-YINC was associated with shorter overall survival of GBM patients. Functionally, lnc- YINC acted in-trans and was a key regulator of cell cycle progression of GBM cells/glioma stem cells (GSCs) and self-renewal of GSCs. It also protected GBM cells/GSCs from DNA double-strand breaks (DSBs) caused by endogenous stress or exogenous ionizing radiation. Mechanistically, lnc-YINC interacted with YBX1, and post-transcriptionally regulated the expression of key regulators of cell cycle or DSB repair, at least partially by stabilizing their mRNAs. Based on these new findings, we hypothesize that lnc-YINC is a GBM-promoting lncRNA that promotes proliferation of and modulates radio-sensitivity of GBM cells/GSCs, by enhancing YBX1 binding to key regulators of cell cycle/DSB repair and co-regulating their expression at post-transcriptional level. The objectives of this proposal are (a) to determine the role of lnc-YINC in gliomagenesis, (b) to integrate enhanced CLIP-seq (eCLIP-seq), ribosome profiling, RNA-seq and quantitative mass spectrometry data, with functional assays to identify the downstream targets of lnc-YINC/YBX1 that are key to regulating cell cycle or DSB repair, (c) to dissect the molecular mechanisms whereby lnc-YINC/YBX1 axis post-transcriptionally regulates the expression of key regulators of cell cycle or DSB repair, (d) to determine the role of lnc-YINC in modulating radio-sensitivity of GBM cells/GSCs and to investigate the therapeutic impact of combining inhibition of lnc-YINC with radiation therapy on GBM maintenance in vivo. This application is strengthened by a team of experts of basic/clinical science of GBM, DNA damage repair, RNA biology and proteomics.