Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation

Genome Med. 2020 Oct 22;12(1):89. doi: 10.1186/s13073-020-00788-5.

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 Wnt-addicted 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 co-expression 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: CRISPRi screen; Cancer; Functional lncRNAs; Wnt signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Computational Biology / methods
  • Gene Editing
  • Gene Expression Profiling* / methods
  • Gene Expression Regulation*
  • Gene Expression Regulation, Neoplastic
  • Gene Knockdown Techniques
  • Gene Regulatory Networks
  • Heterografts
  • Humans
  • Mice
  • Neoplasms / genetics
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Open Reading Frames
  • Quantitative Trait Loci
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Long Noncoding*
  • Wnt Proteins / metabolism*
  • Wnt Signaling Pathway

Substances

  • RNA, Long Noncoding
  • Wnt Proteins

Associated data

  • figshare/10.6084/m9.figshare.13012535