Transcribed Ultra Conserved Regions in Glioblastoma

摘要 胶质母细胞瘤(GBM)是最常见、最致命的原发脑肿瘤。大多数GBM研究都有 重点是蛋白质编码基因，而较少研究非编码转录本，这些转录本占细胞RNA的98%。 转录的超保守区(TUCRs)是一组100%保守的481个转录本 多种物种。它们对变异具有高度的抵抗力，在癌症中通常被解除调控，这表明 监管和功能的重要性。一些证据表明，大多数TUCR是长的非编码RNA (LncRNAs)高度保守，不像大多数其他通常保守程度较低的lncRNAs。TUCR是 在癌症方面的研究很少，而在GBM方面则完全没有。截至提交本R21申请之日起，有 目前还没有关于GBM中TUCRs的研究发表。在前期工作中，我们进行了TUCR的第一次分析 使用癌症基因组图谱(TCGA)RNA-Seq数据在GBM中表达，并鉴定出194个TUCR 相对于正常脑的差异表达。这些TUCR中的许多与患者的生存相关。这 该项目旨在识别和表征在GBM中差异表达的TUCR，并揭示其 作用和作用机制。我们提出了三个具体目标。在目标1中，我们将确定和 描述在GBM中差异表达的TUCR的特征。候选TUCR将从以下位置确定 TCGA RNA-Seq数据根据它们的差异表达和与生存的相关性进行分析和优先排序。 他们的完整转录序列将被发现，他们的lncRNA状态将得到验证。在《目标2》中，我们将揭开 选择TUCR基因在GBM中的作用。将使用来自AIM 1的排名前20的TUCR lncRNA 为了这个目的。在GBM细胞系和干细胞中，TUCR lncRNAs会过表达并被敲除。这个 TUCRs对细胞增殖、存活、侵袭、迁移和干细胞分化的影响 将分析RCAS/TVA免疫活性的GBM小鼠模型的体内肿瘤生长情况。在目标3中，我们 将确定在GBM中选择TUCR lncRNA的作用机制。发挥监管作用的TUCR 对于目标2中确定的基底膜恶性肿瘤，将优先考虑。我们将确定每个细胞的亚细胞定位 TUCR lncRNA和使用由生物信息学和实验测定组成的多管齐下的方法 蛋白质和核酸结合伙伴随后进行功能抢救实验以揭示其机制 TUCR lncRNA的作用。该项目的成功完成将代表着第一个全面的 分析基底膜中的TUCRs，并产生关于基底膜恶性机制的新知识。

ABSTRACT Glioblastoma (GBM) is the most common and deadliest primary malignant brain tumor. Most GBM research has focused on protein-coding genes and less on non-coding transcripts that make up 98% of cellular RNA. Transcribed Ultra-Conserved Regions (TUCRs) are a group of 481 transcripts that are 100% conserved across multiple species. They are highly resistant to variation and are commonly deregulated in cancer, suggesting regulatory and functional importance. Some evidence suggests that most TUCRs are long non-coding RNAs (lncRNAs) that are highly conserved, unlike most other lncRNAs that are usually poorly conserved. TUCRs are largely understudied in cancer and not at all in GBM. As of the date of submission of this R21 application, there were no published studies on TUCRs in GBM. In preliminary work, we performed the first analysis of TUCR expression in GBM using The Cancer Genome Atlas (TCGA) RNA-Seq data and identified 194 TUCRs that are differentially expressed relative to normal brain. Many of these TUCRs correlated with patient survival. This project aims to identify and characterize TUCRs that are differentially expressed in GBM and to uncover their functions and mechanisms of action. We propose three specific aims. In Aim 1, we will identify and characterize TUCRs that are differentially expressed in GBM. Candidate TUCRs will be identified from TCGA RNA-Seq data analyses and prioritized based on their differential expression and correlation with survival. Their full transcript sequences will be uncovered and their lncRNA status verified. In Aim 2, we will uncover the functions of select TUCR lncRNAs in GBM. The top 20 ranked TUCR lncRNAs from aim 1 will be used for this aim. TUCR lncRNAs will be overexpressed and knocked down/out in GBM cell lines and stem cells. The effects of the TUCRs on cell proliferation, survival, invasion, migration and stem cell differentiation as well as on in vivo tumor growth in an RCAS/Tva immune competent mouse model of GBM will be analyzed. In Aim 3, we will identify the mechanism of action for select TUCR lncRNAs in GBM. TUCRs that exert regulatory effects on GBM malignancy as determined in aim 2 will be prioritized. We will identify the subcellular localization of each TUCR lncRNA and use a multipronged approach consisting of bioinformatics and experimental determination of protein and nucleic acid binding partners followed by functional rescue experiments to uncover the mechanisms of action of TUCR lncRNAs. Successful completion of this project would represent the first comprehensive analysis of TUCRs in GBM and generate new knowledge on the mechanisms of GBM malignancy.