Date: Monday, November 6, 2017
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Long noncoding RNAs (lncRNAs) are a class of transcripts regulating gene expression. We have recently identified a novel lncRNA, H19X, which was upregulated in the skin of patients with SSc. We also demonstrated that TGFβ regulates H19X expression and is a key mediator of myofibroblast development and extracellular matrix synthesis. Here we aimed to assess whether (1) H19X is a general regulator of TGFβ-driven fibrotic diseases and (2) H19X upregulation in SSc dermal fibroblasts is anti-apoptotic and pro-proliferative thereby favoring fibrosis.
Methods: To study the function of H19X in apoptosis and proliferation of dermal fibroblasts we silenced H19X using locked nucleic acid oligonucleotides (LNA GapmeRs) followed by microarray analysis, qPCR, BrdU cell proliferation assay, Caspase 3/7 apoptosis assay and scratch assay. Cells were treated with 10 ng/ml TGFβ. Lung tissues were obtained from patients with SSc and idiopathic pulmonary fibrosis (IPF) undergoing organ transplantation, and from healthy controls (HC). Fibrotic gut tissues were obtained from patients with Crohn’s disease undergoing gut resection. Expression of H19X was analyzed by qPCR.
Results: H19X expression was significantly increased in SSc interstitial lung disease and IPF patients versus HC (n=11 each, p<0.05). A significant H19X overexpression was also detected in fibrotic gut tissue from patients with Crohn’s disease versus control (n=10 and 4 respectively; p<0.05). Furthermore, H19X was found to be increased in physiological wound healing tissue versus HC samples (n=11 and 8 respectively; p<0.05). Upregulation of H19X was paralleled by an upregulation of PAI-1, indicating TGFβ pathway activation in these tissues.
H19X knockdown followed by microarray analysis (n=5) showed that “FAS signaling pathway”, “cyclins and cell cycle regulation”, “regulation of cell cycle progression by Plk3”, and “free radical induced apoptosis” were among the pathways with the highest number of significantly enriched genes (p<0.005). Apoptosis markers like BCL2, IGFBP3 and FAF1 (n=5, p<0.05) were significantly downregulated in H19X-silenced fibroblasts. These results indicated that targeting H19X might have a pro-apoptotic effect on fibroblasts. Functional studies of apoptosis confirmed enhanced fibroblast apoptosis after H19X silencing and TGFβ stimulation (n=5, p<0.05). In addition to decreased apoptosis, increased fibroblast proliferation might also favor fibrosis. Indeed, H19X downregulation led to reduced fibroblast proliferation as measured by BrdU assay (n=5, p<0.05). Scratch assays (n=5) showed that H19X knockdown decreased TGFβ reduced wound healing.
Conclusion: H19X supports TGFβ-driven fibrosis by inducing proliferation and reducing apoptosis of fibroblasts. These effects are not limited to SSc, but also apply to a wider range of fibrotic diseases. Our results highlight the role of the novel lncRNA H19X as an important profibrotic mediator in TGFβ mediated processes.
To cite this abstract in AMA style:Pachera E, Wunderlin A, Assassi S, Salazar G, Frank Bertoncelj M, Dobrota R, Brock M, Feghali-Bostwick CA, Dijkstra G, Rogler G, van Haaften Wouter T, Distler J, Kania G, Distler O. Long Noncoding RNA H19X Is a Key Regulator of Apoptosis and Proliferation of Fibroblasts in Systemic Sclerosis and Other TGFβ-Driven Fibrotic Diseases [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/long-noncoding-rna-h19x-is-a-key-regulator-of-apoptosis-and-proliferation-of-fibroblasts-in-systemic-sclerosis-and-other-tgf%ce%b2-driven-fibrotic-diseases/. Accessed September 25, 2021.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/long-noncoding-rna-h19x-is-a-key-regulator-of-apoptosis-and-proliferation-of-fibroblasts-in-systemic-sclerosis-and-other-tgf%ce%b2-driven-fibrotic-diseases/