Background/Purpose: Long noncoding RNAs (LncRNAs) are emerging as a novel class of noncoding transcripts involved in the regulation of gene expression. So far, for only few of them the function has been elucidated therefore their role in disease is poorly understood.

Methods: Here we aimed to identify candidate lncRNAs in systemic sclerosis (SSc) and investigate their function, particularly in relation to the TGFβ pathway and the development of the myofibroblast phenotype. RNA Sequencing Ilumina HiSeq2000 was performed in healthy control (HC) and SSc skin biopsies. Human skin fibroblasts were isolated from biopsies of SSc patients and HC; human pulmonary smooth muscle cells were purchased from Lonza. Cells were treated with 10 ng/ml TGFβ. TGFβR1 inhibitors (SD208 and SB431542) and siRNA against SMAD3 and SMAD4 were used to investigate TGFβ driven gene expression. LncRNA H19X was silenced in skin fibroblasts using locked nucleic acid antisense oligonucleotides (LNA GapmeRs), followed by qPCR analyses, immunofluorescence staining and Sircol assay. The expression of H19X was also measured in tissue samples of liver and lung fibrosis.

Results: RNA sequencing showed a significant upregulation of the lncRNA H19X in SSc versus HC skin biopsies. Importantly, the upregulation of H19X was not limited to SSc skin, but present also in the tissues from liver and lung fibrosis, indicating a broader role of H19X in fibrotic diseases. While there was no difference in the basal expression of H19X between SSc and HC cultured dermal fibroblasts, H19X was strongly and consistently induced by TGFβ. Induction of H19X by TGFβ was not limited to skin fibroblasts, but evident also in other cell types relevant for SSc, e.g. pulmonary vascular smooth muscle cells. Time curve analysis revealed that the upregulation of H19X by TGFβ was strongest after 6h reaching 12.8±0.7 induction; and dose curve analysis showed a steady increase of H19X over physiologically relevant TGFβ concentrations. These effects were TGFβR1 dependent as shown by the inhibition experiments with the chemical inhibitors SD208 and SB431542. Moreover, the upregulation of H19X by TGFβ was significantly impaired by silencing of SMAD3 and SMAD4, further pointing to a role of the canonical TGFβ pathway in H19X induction. Knockdown of H19X in skin fibroblasts led to a strong down-regulation of pro-fibrotic genes COL1A1, fibronectin and αSMA mRNAs. Immunofluorescence staining showed reduced αSMA and stress fibers formation in H19X silenced dermal fibroblasts indicating an involvement of H19X in the development of a myofibroblast phenotype. Furthermore, Sircol assay revealed a decrease of total collagen secretion upon H19X knockdown confirming the pro-fibrotic effects of H19X. 

Conclusion:  This is the first study reporting changes in long-non coding RNAs in SSc and across fibrotic diseases. It opens new perspectives in studying the pathogenesis of fibrotic diseases by this novel class of regulatory, non-coding RNAs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/long-noncoding-h19x-is-a-key-mediator-of-tgf-beta-induced-pro-fibrotic-effects-in-the-pathogenesis-of-systemic-sclerosis-and-other-fibrotic-diseases/