Session Type: ACR Concurrent Abstract Session
Session Time: 4:30PM-6:00PM
Background/Purpose: Long noncoding RNAs (lncRNAs) are an emerging class of large noncoding transcripts involved in the regulation of gene expression in health and disease. We have recently identified a novel lncRNA, H19X, which is strictly regulated by TGFβ in a dose and time dependent manner. Here we aim to characterize the expression of H19X in Systemic Sclerosis (SSc) and other fibrotic diseases as well as its function in fibrotic events.
Methods: Skin and lung biopsies from patients with SSc, idiopathic pulmonary fibrosis (IPF), and healthy controls (HC) were obtained from cohorts at four different expert centers. Expression of H19X was analyzed by RNA Sequencing Ilumina HiSeq2000 and quantitative (q)PCR respectively. The function of H19X was investigated in skin fibroblasts transfected with locked nucleic acid oligonucleotides (LNA GapmeRs) by using the following methods: microarray analysis, qPCR, immunofluorescence, sircol, contraction assay, ELISA and Western blot (WB). In situ hybridization of H19X in SSc dermal fibroblast was performed using Stellaris FISH probes.
Results: H19X expression was consistently upregulated across all the four cohorts (SSc n=34, HC= 26), using different techniques. The upregulation was also consistent across different subsets of patients and between patients with different disease durations. H19X showed a similar expression profile in clinically non-fibrotic and clinically fibrotic skin biopsies indicating a role in early disease development. Moreover, H19X expression was also significantly increased in SSc interstitial lung disease patients versus HC (n=11 each, p<0.05). A significant H19X overexpression was also detected in IPF samples suggesting a broader role of H19X in fibrotic diseases (n=11 each, p<0.05). Microarray analysis after H19X silencing revealed a strong involvement in extracellular matrix production with collagens being the most downregulated genes. Accordingly, collagen catabolic process, extracellular matrix organization and extracellular matrix disassembly were among the pathways with highest number of enriched genes. Downregulation of collagen Iα1, fibronectin and α-smooth muscle actin (αSMA) after H19X knockdown was confirmed by qPCR (n=5, p<0.05). Sircol assay for pan-collagen production, ELISA for pro-collagen Iα1 and WB analysis for fibronectin confirmed the importance of H19X in the regulation of extracellular matrix components. Additionally, silencing of H19X significantly impaired αSMA fiber formation, stress fiber formation as well as cell contractility strongly suggesting an important role of H19X in the development of the myofibroblast phenotype (n=5-6, p<0.05). Cell fractionation showed that TGFβ induced expression of H19X is localized mainly into the nucleus. In situ hybridization confirmed H19X localization as mainly nuclear and within a defined spot indicating that H19X could influence gene expression by interacting directly with the chromatin (n=4).
Conclusion: This is the first study reporting a significant upregulation of H19X expression in SSc and across fibrotic organs. By focusing on this novel class of regulatory, noncoding RNAs new perspectives in the pathogenesis of fibrotic diseases are opened.
To cite this abstract in AMA style:Pachera E, Assassi S, Salazar G, Frank Bertoncelj M, Dobrota R, Brock M, Kurreeman F, de Vries-Bouwstra JK, Messemaker T, Feghali-Bostwick C, Distler JH, Kania G, Distler O. Long Noncoding RNA H19X Is a Master Regulator of Extracellular Matrix Production in Systemic Sclerosis and Other Fibrotic Disease [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/long-noncoding-rna-h19x-is-a-master-regulator-of-extracellular-matrix-production-in-systemic-sclerosis-and-other-fibrotic-disease/. Accessed November 18, 2019.
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