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

系统解析长非编码RNA在胶质母细胞瘤中的功能和机制

• 批准号: 10208228
• 负责人: Yiwen Chen
• 金额: $ 40.5万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10208228
• 关键词: Adult; Binding; Biological Assay; Biology; Brain; CCND1 gene; CCNE2 gene; CRISPR interference; Cell Cycle; Cell Cycle Progression; Cell Proliferation; Cells; Clinical; Clinical Sciences; Code; DNA Double Strand Break; DNA Repair; Data; Development; Diagnostic; Diffuse; Disease; Dissection; Double Strand Break Repair; E2F transcription factors; FOXM1 gene; G1/S Transition; Genes; Genetic Transcription; Genomics; Glioblastoma; Glioma; Gliomagenesis; Goals; Human; In Vitro; Infiltration; Ionizing radiation; Maintenance; Malignant - descriptor; Mass Spectrum Analysis; Mediating; Messenger RNA; Molecular; Neuroglia; Nucleotides; Pathogenesis; Patients; Post-Transcriptional Regulation; Primary Brain Neoplasms; Prognosis; Prognostic Marker; Proteins; Proteomics; RNA; RNA-Binding Proteins; Radiation Tolerance; Radiation therapy; Regulation; Research; Resistance; Role; Spinal Cord Neoplasms; Stress; Therapeutic; Untranslated RNA; Xenograft Model; base; brain tissue; cell growth; crosslinking and immunoprecipitation sequencing; effective therapy; gain of function; homologous recombination; in vivo; insight; loss of function; multiple omics; novel; novel therapeutic intervention; protein complex; public health relevance; ribosome profiling; self-renewal; standard care; stem cell proliferation; stem cell self renewal; stem cells; therapeutically effective; transcription factor; transcriptome sequencing; tumor

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.

项目摘要/摘要 胶质母细胞瘤（GBM）是一种高级神经胶质瘤（IV级），是最普遍，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。经过标准处理后，GBM的中位存活率 患者大约15个月。 GBM以增强的细胞增殖，高倾向的特征 整个大脑的侵袭/弥漫性浸润以及对化学/辐射疗法的抗性。新兴 有证据支持长的非编码RNA（LNCRNA，约18,000个人lncRNA基因）可以介导肿瘤 促进/抑制效果并充当独立的诊断/预后生物标志物。我们的先前 综合基因组研究表明，许多LNCRNA表现失调，与 临床预后，或携带GBM中频繁的躯体拷贝数改变，这表明 GBM发病机理中的LNCRNA。但是，GBM中大多数LNCRNA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摩变方法来表征该功能 LNCRNA在GBM中的机制，并帮助基于新颖见解的新治疗策略来开发新的治疗策略 进入GBM中的lncRNA调节。我们使用CRISPR干扰（CRISPRI）的大规模功能丢失屏幕 确定了反义lncRNA，lnc-yinc（ybx1相互作用的lncRNA），这对于GBM细胞生长和 与正常的脑组织和低度神经胶质瘤（LGG）相比，GBM中的明显上调。这 LNC-yinc的较高表达与GBM患者的总体存活率较短有关。在功能上，LNC- YINC的作用是内部的，是GBM细胞/神经胶质瘤干细胞（GSC）细胞周期进程的关键调节剂（GSC） 和GSC的自我更新。它还保护了GBM细胞/GSC免受DNA双链断裂（DSB）引起的 通过内源性应力或外源性电离辐射。从机械上讲，LNC-yinc与YBX1相互作用，并且 转录后调节细胞周期或DSB修复的关键调节剂的表达，至少部分由 稳定他们的mRNA。基于这些新发现，我们假设LNC-yinc是GBM促进的 通过增强YBX1的lncRNA，促进GBM细胞/GSC的无线电敏感性的增殖和调节 与细胞周期/DSB修复的关键调节剂结合并在转录后水平共同调节其表达。 该提案的目标是（a）确定LNC-yinc在神经胶质作用中的作用，（b）整合 具有增强的夹式（Eclip-Seq），核糖体分析，RNA-SEQ和定量质谱数据，并带有带有的质谱数据 识别LNC-YINC/YBX1的下游目标的功能测定，这是调节细胞周期或 DSB修复，（c）剖析分子机制，在转录后LNC-YINC/YBX1轴 调节细胞周期或DSB修复的关键调节剂的表达，（d）确定LNC-yinc在 调节GBM单元/GSC的无线电敏感性，并研究组合的治疗影响 通过放射治疗在体内维持GBM维护上对LNC-yinc抑制。该应用程序通过 GBM基础/临床科学专家团队，DNA损伤修复，RNA生物学和蛋白质组学。