The lncRNA HOTAIR/EZH2 interaction inhibitor AC1Q3QWB (AQB) attenuates fibrotic SSc skin tissue re-modelling

ObjectivesThe long non-coding RNA HOTAIR has previously been shown to drive pro-fibrotic gene expression in SSc dermal fibroblasts through its ability to interact with EZH2. Targeting EZH2 enzymatic activity can reverse HOTAIR mediated pro-fibrotic gene expression but its many functions make it an undesirable therapeutic target for SSc. Recently inhibitors selectively targeting the HOTAIR/EZH2 interaction have been developed. The aim of this study was to characterise the ability of one of these inhibitors to modulate SSc tissue remodelling. MethodsExplanted healthy and SSc dermal fibroblasts were treated with the HOTAIR/EZH2 inhibitor AC1Q3QWB (AQB) (20{micro}M) for 48 hours. In addition, healthy dermal fibroblasts were transduced with a lentivirus encoding HOTAIR or a scrambled control. Conditioned media from healthy, SSc and HOTAIR-expressing dermal fibroblasts was used to stimulate human keratinocytes (HaCaTs). Scramble control and HOTAIR expressing fibroblasts were grown in 3D skin equivalents containing primary keratinocytes and keratin 9 (K9) immunohistochemistry performed. ResultsAQB inhibits pro-fibrotic gene expression in HOTAIR expressing dermal fibroblasts, validating the specificity of the inhibitor. In SSc patient dermal fibroblasts, AQB blocked pro-fibrotic gene expression but did not affect gene expression in healthy dermal fibroblasts. SSc patient skin was shown to express high levels of the palmoplantar specific K9 and Epithelial to Mesenchymal transition (EMT) markers. Through co-culture experiments we showed these effects were mediated by SSc dermal fibroblasts. This tissue remodelling was disrupted when HOTAIR/EZH2 interaction was inhibited in the fibroblasts with AQB. ConclusionsWe have shown for the first time that directly inhibiting HOTAIR/EZH2 interaction blocks pro-fibrotic gene expression in SSc fibroblasts and tissue re-modelling found in SSc patient skin. This may represent a novel therapeutic intervention.