The human genome produces many thousands of long non-coding RNAs (lncRNAs) ? transcripts >200 nucleotides long with little protein coding potential. It is now clear that lncRNAs can have critical biological functions and roles in human disease including cancer. Because lncRNAs are particularly cell type and disease specific, they are attractive as therapeutic targets. However, our understanding of lncRNAs in primary brain tumors and the normal human brain is still very limited. Glioblastoma (GBM) is the most common primary malignant brain tumor. Despite surgery, chemotherapy and radiation treatment, the median survival after diagnosis of GBM is only 14-16 months. In recent work, my mentor and local colleagues developed CRISPRi for large-scale, genome-wide screening of lncRNA function in human cells. From this screen of 16,401 lncRNAs across seven human cell types ? including GBM cells, induced pluripotent stem cells, and multiple cancer lines ? they uncovered hundreds of lncRNAs that regulate proliferation, with 65 ?hits? in U87 GBM cells. Knockdown of one particular lncRNA ? LINC00263 ? strongly reduced glioma cell propagation in U87 cells and two patient-derived primary glioma lines, not in other cell types assayed. This glioma-specific lethality of LINC00263 depletion is particularly intriguing as this lncRNA is expressed in all analyzed cell types, suggesting that certain lncRNAs can be selectively ?wired? into essential cellular processes or that certain cell types exhibit synthetic sensitivity to LINC00263 loss. Additionally, by single-molecule fluorescent in situ hybridization, LINC00263 transcripts localized exclusively to the nucleus. Nuclear lncRNAs can regulate gene expression through interaction with chromatin regulators, and public RNA Immunoprecipitation Sequencing (RIP-Seq) data reveal significant interactions between LINC00263 and multiple repressive chromatin modifiers, including SUZ12 and HDAC1. Given these Preliminary Studies, my central hypothesis is that LINC00263 regulates essential gene expression through chromatin-modifying complexes in a cell type-specific manner. In this proposal, my first Aim is to determine the role of LINC00263 in models of human GBM and normal neural and glial cell types both in vitro and in vivo, including in primary patient samples. For my second Aim, I will more comprehensively identify and compare the protein interactors of LINC00263 in multiple cell types. In addition to my mentor?s expertise with lncRNAs and Preliminary Studies, our ongoing local collaborations with Dr. Jonathan Weissman (CRISPRi and CRISPRa), Dr. Aaron Diaz (bioinformatics), and Dr. Theo Nicolaides (primary samples and in vivo models) support the feasibility of this work. Many fundamentally important discoveries have been made through studying individual lncRNAs, and despite the surge in descriptive atlases of lncRNA expression, there is still relatively little understanding of how lncRNA function in the cell, both normally and in disease. By accomplishing these Aims, I will lay novel, important groundwork for understanding the roles, mechanisms, and cell type-specific function of an important lncRNA in human neurologic disease.
The human genome encodes tens of thousands of long non-coding RNAs (lncRNAs), and specific lncRNAs may play key roles or serve as therapeutic targets in human neurologic diseases including glioblastoma. LINC00263 is an uncharacterized lncRNA we found to be essential for the growth of multiple glioma cell lines but not in several other cell types. This work will characterize the function of LINC00263 in human glioma as well as normal brain cells and determine how it works on a molecular level, providing a framework for understanding lncRNAs that are selectively important for glioma proliferation.
