Design and Functional Validation of a Mutant Variant of the LncRNA <i>HOTAIR</i> to Counteract Snail Function in Epithelial-to-Mesenchymal Transition

Abstract

<i>HOTAIR</i> is a lncRNA overexpressed in several epithelial cancers and strongly correlated with invasion. This lncRNA was proven a pivotal element of the epithelial-to-mesenchymal transition (EMT), a transdifferentiation process triggering metastasis. Snail, master inducer of EMT, requires <i>HOTAIR</i> to recruit EZH2 on specific epithelial target genes (i.e., <i>HNF4α, E-cadherin</i>, and <i>HNF1α</i>) and cause their repression. Here, we designed a <i>HOTAIR</i> deletion mutant form, named <i>HOTAIR</i>-<i>sbid</i>, including the putative Snail-binding domain but depleted of the EZH2-binding domain. <i>HOTAIR</i>-<i>sbid</i> acted as a dominant negative of the endogenous <i>HOTAIR</i>. In both murine and human tumor cells, <i>HOTAIR</i>-<i>sbid</i> impaired the ability of <i>HOTAIR</i> to bind Snail and, in turn, trigger H3K27me3/EZH2-mediated repression of Snail epithelial target genes. Notably, <i>HOTAIR</i>-<i>sbid</i> expression was proven to reduce cellular motility, invasiveness, anchorage-independent growth, and responsiveness to TGFβ-induced EMT. These data provide evidence on a lncRNA-based strategy to effectively impair the function of a master EMT-transcriptional factor. SIGNIFICANCE: This study defines an innovative RNA-based strategy to interfere with a pivotal function of the tumor-related lncRNA <i>HOTAIR</i>, comprising a dominant negative mutant that was computationally designed and that impairs epithelial-to-mesenchymal transition.