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

项目摘要/摘要 胶质母细胞瘤(Gbm)是一种高级别(IV级)的胶质瘤，是最常见和最恶性的原发脑肿瘤。 在成年人身上。目前还没有有效的治疗方法。经过标准治疗后，GBM的中位生存期 患者大约15个月。GBM的特点是促进细胞增殖，高度倾向于 侵袭/弥漫性渗透到整个大脑，以及对化疗/放射治疗的抵抗。新兴 有证据表明，长的非编码RNA(lncRNAs，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 综合基因组学研究显示，许多lncRNAs表现出表达异常，与 临床预后，或存在频繁的GBM体细胞拷贝数改变，提示 LncRNAs在GBM发病机制中的作用然而，大多数lncRNAs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化的多组学方法来表征功能 以及在GBM中lncRNAs的作用机制，并基于新的洞察力帮助开发新的治疗策略 转化为GBM中的lncRNA调控。我们使用CRISPR干扰的大型功能丧失屏幕(CRISPRi) 发现了一种反义lncRNA，lnc-YINC(YBX1-相互作用的lncRNA)，它对GBM细胞的生长和 与正常脑组织和低级别胶质瘤(LGG)相比，GBM中表达显著上调。这个 LNC-YINC的高表达与GBM患者的总生存期缩短有关。在功能上，lnc- 银杏叶提取物是GBM细胞/胶质瘤干细胞(GSCs)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/GSCs免受DNA双链断裂(DSB)的影响 通过内源应激或外源电离辐射。从机制上讲，LNC-YINC与YBX1相互作用，并且 转录后调控细胞周期或DSB修复的关键调节因子的表达，至少部分是通过 稳定他们的核糖核酸。基于这些新的发现，我们假设LNC-YINC是一种GBM促进因子 通过增强YBX1促进GBM细胞/GSCs增殖并调节其放射敏感性的lncRNA 结合细胞周期/DSB修复的关键调节因子，并在转录后水平共同调节它们的表达。 这项提案的目标是(A)确定lnc-YINC在胶质瘤发生中的作用，(B)整合 增强的CLIP-SEQ(eCLIP-SEQ)、核糖体图谱、RNA-SEQ和定量质谱学数据， 确定LNC-YINC/YBX1下游靶点的功能分析，这些下游靶点是调节细胞周期或 DSB修复，(C)剖析lnc-YINC/YBX1转录后轴的分子机制 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/GSCs的放射敏感性及联合应用的疗效观察 放射治疗对银杏叶提取物体内GBM维持的抑制作用。这个应用程序通过一个 GBM、DNA损伤修复、RNA生物学和蛋白质组学的基础/临床科学专家团队。

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.