Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation
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RESEARCH
Open Access
Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation Shiyang Liu1,2, Nathan Harmston3, Trudy Lee Glaser1, Yunka Wong1, Zheng Zhong1, Babita Madan1, David M. Virshup1,4* and Enrico Petretto2,5*
Abstract Background: Wnt signaling is an evolutionarily conserved developmental pathway that is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not been systematically characterized. Methods: We comprehensively mapped Wnt-regulated lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model and examined the response of lncRNAs to Wnt inhibition between in vivo and in vitro cancer models. We further annotated and characterized these Wnt-regulated lncRNAs using existing genomic classifications (using data from FANTOM5) in the context of Wnt signaling and inferred their role in cancer pathogenesis (using GWAS and expression data from the TCGA). To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation both in vivo and in vitro. Results: We identified 3633 lncRNAs, of which 1503 were regulated by Wnt signaling in an orthotopic Wntaddicted pancreatic cancer model. These lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. Our analysis suggested that Wnt signaling inhibition could influence the coexpression relationship of Wnt-regulated lncRNAs and their eQTL-linked protein-coding genes. Wnt-regulated lncRNAs were also implicated in specific gene networks involved in distinct biological processes that contribute to the pathogenesis of cancers. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1503) of the Wnt-regulated lncRNAs were found to modify cancer cell growth in vitro. This included CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-regulated lncRNAs (25/1503) that had a functional effect on cancer cell growth. Conclusions: Our study demonstrates the value of studying lncRNA functions in vivo, provides a valuable resource of lncRNAs regulated by Wnt signaling, and establishes a framework for systematic discovery of functional lncRNAs. Keywords: Functional lncRNAs, Wnt signaling, Cancer, CRISPRi screen
* Correspondence: [email protected]; [email protected] 1 Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore 2 Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to
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