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

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.

In silico development and experimental validation of a novel 7-gene signature based on PI3K pathway-related genes in bladder cancer

“…CDK6 overexpression has been found in several cancers, and tumor drug resistance increases when CDK6 expression is elevated (Tadesse et al 2015;Yang et al 2017;Li et al 2018;Cornell et al 2019). Upregulation of ODK6 promotes the proliferation of colon cancer (Liu et al 2020), breast cancer (Leung (Feng et al 2017). ITGB7 is involved in regulating adhesion (Ohguchi et al 2016) and proliferation and glucose metabolism in cervical cancer (Chai et al 2019).…”

Bioinformatics analysis of lncRNAs in the occurrence and development of osteosarcoma

“…Studies have shown that OLMALINC (LINC00263), regulated by heterogeneous nuclear ribonucleoprotein K, upregulates CAPN2 through miR-147a, and thus plays an important role in cancer and malignant tumors ( 40 ). Additionally, Wnt-regulated OLMALINC (LINC00263) was found to modify cancer cell growth ( 41 ). This is consistent with our results, but the specific mechanism of action needs to be further studied through a large number of animal experiments.…”

Microdeletion of 9q22.3: A patient with minimal deletion size associated with a severe phenotype

“…Loci containing let-7 family microRNAs are frequently within 9q22.3 deletions and have been implicated in aspects of the phenotype (Yamada et al, 2020) but these sequences, at around 9:94,220,000, are outside the deletion in the present patient. A recent article (Liu et al, 2020) showed that a sequence within the deletion (designated RP11-43505.2 in the article, which maps close to MT1P1…”

“…Loci containing let‐7 family microRNAs are frequently within 9q22.3 deletions and have been implicated in aspects of the phenotype (Yamada et al, 2020) but these sequences, at around 9:94,220,000, are outside the deletion in the present patient. A recent article (Liu et al, 2020) showed that a sequence within the deletion (designated RP11‐43505.2 in the article, which maps close to MT1P1 and likely overlaps with FANCC‐203 ) is targeted by WNT signaling. Several WNT genes are targets of hedgehog signaling (Onodera et al, 2017), and their expression would likely be increased when one copy of PTCH1 is deleted.…”