CRISPRi screen of long non-coding RNAs identifies LINC03045 regulating glioblastoma invasion.

Autor: Tsung K; Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Liu KQ; Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Han JS; Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Deshpande K; Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Doan T; Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Loh YE; USC Libraries Bioinformatics Services, University of Southern California, Los Angeles, California, United States of America., Ding L; Department of Preventative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Yang W; Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Neman J; Department of Neurological Surgery, Physiology and Neuroscience, USC Brain Tumor Center, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Dou Y; Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.; Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America., Attenello FJ; Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.; Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
Jazyk: angličtina
Zdroj: PLoS genetics [PLoS Genet] 2024 Jun 10; Vol. 20 (6), pp. e1011314. Date of Electronic Publication: 2024 Jun 10 (Print Publication: 2024).
DOI: 10.1371/journal.pgen.1011314
Abstrakt: Introduction: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates.
Methods: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments.
Results: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD.
Conclusion: CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2024 Tsung et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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