Transcribed Ultra Conserved Regions in Glioblastoma

摘要 胶质母细胞瘤（GBM）是最常见和最致命的原发性恶性脑肿瘤。大多数GBM研究 他们专注于蛋白质编码基因，而较少关注占细胞RNA 98%的非编码转录物。 转录的超保守区（TUCR）是一组481个转录物，它们在整个基因组中是100%保守的。 多个物种它们对变异具有高度抵抗力，并且在癌症中通常是失调的，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， 这些lncRNA是高度保守的，不像大多数其他lncRNA通常保守性很差。TUCR是 在癌症中研究不足，而在GBM中则完全没有。截至提交本R21申请之日， 没有发表关于GBM中TUCR的研究。在前期工作中，我们对TUCR进行了首次分析 使用癌症基因组图谱（TCGA）RNA-Seq数据在GBM中表达，并鉴定了194个TUCR， 相对于正常脑的差异表达。这些TUCR中的许多与患者生存相关。这 该项目旨在鉴定和表征GBM中差异表达的TUCR，并揭示其 作用和机制。我们提出三个具体目标。在目标1中，我们将确定和 表征在GBM中差异表达的TUCR。候选TUCR将从 TCGA RNA-Seq数据分析并根据其差异表达和与生存的相关性进行优先排序。 它们的完整转录物序列将被发现，它们的lncRNA状态将被验证。在目标2中，我们将发现 选择TUCR lncRNA在GBM中的功能。将使用来自aim 1的排名前20位的TUCR lncRNA 为了这个目的。TUCR lncRNA将在GBM细胞系和干细胞中过表达并被敲低/敲除。的 TUCR对细胞增殖、存活、侵袭、迁移和干细胞分化的影响以及对 将分析GBM的RCAS/Tva免疫活性小鼠模型中的体内肿瘤生长。在目标3中， 将确定选择TUCR lncRNA在GBM中的作用机制。发挥调节作用的TUCR 将优先考虑目标2中确定的GBM恶性肿瘤。我们将确定每一个的亚细胞定位 TUCR lncRNA，并使用生物信息学和实验测定组成的多管齐下的方法， 蛋白质和核酸结合伴侣，随后进行功能性拯救实验以揭示机制 TUCR lncRNA的作用。该项目的成功完成将是第一个全面的 GBM中TUCR的分析，并产生关于GBM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.