Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore.
Program in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore.
Genome Med. 2020 Oct 22;12(1):89. doi: 10.1186/s13073-020-00788-5.
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.
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.
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.
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.
Wnt 信号是一种进化上保守的发育途径，在癌症中经常被过度激活。虽然已知有多个受 Wnt 信号调控的蛋白质编码基因，但受 Wnt 信号调控的功能性长链非编码 RNA（lncRNA）尚未得到系统表征。
我们从一个原位依赖 Wnt 的胰腺癌模型中全面绘制了受 Wnt 调控的 lncRNA，并在体内和体外癌症模型中检查了 lncRNA 对 Wnt 抑制的反应。我们进一步使用现有的基因组分类（使用 FANTOM5 数据）在 Wnt 信号的背景下注释和表征这些受 Wnt 调控的 lncRNA，并推断它们在癌症发病机制中的作用（使用 GWAS 和 TCGA 的表达数据）。为了功能验证受 Wnt 调控的 lncRNA，我们进行了 CRISPRi 筛选，以评估它们在体内和体外对癌细胞增殖的作用。
我们鉴定了 3633 个 lncRNA，其中 1503 个在原位依赖 Wnt 的胰腺癌模型中受 Wnt 信号调控。这些 lncRNA 对外源移植瘤中 Wnt 信号变化的敏感性远高于培养细胞。我们的分析表明，Wnt 信号抑制可能会影响受 Wnt 调控的 lncRNA 与其 eQTL 连接的蛋白质编码基因之间的共表达关系。受 Wnt 调控的 lncRNA 还与特定的基因网络有关，这些基因网络参与了不同的生物学过程，有助于癌症的发病机制。与之前的全基因组 lncRNA CRISPRi 筛选一致，约 1%（13/1503）的受 Wnt 调控的 lncRNA 被发现可在体外调节癌细胞生长。这包括之前报道的调节癌症生长的 CCAT1 和 LINC00263。通过体内 CRISPRi 筛选，我们将发现的受 Wnt 调控的 lncRNA 的数量增加了一倍（25/1503），这些 lncRNA 对癌细胞生长有功能影响。
我们的研究证明了在体内研究 lncRNA 功能的价值，提供了受 Wnt 信号调控的 lncRNA 的宝贵资源，并为系统发现功能性 lncRNA 建立了框架。
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