Background/Purpose: Recent studies indicate that genes involved in RNA methylation may play a significant role in cellular functions, and disruptions in RNA methylation have been linked to various pathological conditions, including autoimmune diseases. However, its specific role in systemic sclerosis (SSc) remains unclear. RNA methylation influences cellular biological functions by regulating gene expression, particularly through m6A RNA methylation. This reversible process relies on “writer” methyltransferases, “eraser” demethylases, and “reader” proteins that recognize m6A marks. Given the association of m6A marks with fibrosis, we examined the expression of genes involved in m6A RNA methylation by analyzing publicly available datasets and isolated dermal fibroblasts from both healthy controls and SSc patients, further focused on RNA demethylases in SSc fibroblasts.

Methods: Data from GEO was analyzed. RNA was extracted from skin fibroblasts isolated from healthy individuals and SSc patients. Gene expression was analyzed using quantitative PCR. Knockdown or inhibition of ALKBH5 or FTO was done by siRNAs or inhibitors ALKBH5-in-3 and FB23-2. Fibrotic endpoints were examined by various assays. Statistical analyses with a P value below 0.05 were considered statistically significant.

Results: Analysis of the GEO dataset generated in whole skin biopsies revealed a distinct pattern of gene expression. While methylation writers and readers, such as WTAP, METTL14, YTHDC1, and YTHDF2 were significantly downregulated, the eraser demethylases FTO and ALKBH5 were notably upregulated in SSc. This upregulation correlated positively with the modified Rodnan skin score (mRSS). Follow-up quantitative PCR analyses in dermal fibroblasts from healthy controls and patients with diffuse SSc substantiated these findings. Subsequent experiments demonstrated that the knockdown of ALKBH5 or FTO in SSc fibroblasts led to a significant reduction in the expression of pro-fibrotic genes, including collagen genes and TGFβ. Moreover, applying specific inhibitors to these demethylases resulted in marked anti-fibrotic effects, reducing collagen production and fibroblast activation.

Conclusion: Our research elucidates the critical roles of RNA demethylases ALKBH5 and FTO in SSc, particularly in the context of fibrosis. The upregulation of these enzymes and their significant correlation with mRSS suggest they are key drivers in the progression of fibrosis in SSc. Functional experiments demonstrate the potential of targeting ALKBH5 and FTO to reduce pro-fibrotic gene expression and fibroblast activation, offering promising therapeutic avenues for intervention. These findings contribute substantially to our understanding of the molecular underpinnings of SSc and suggest that modulating RNA methylation could be a viable strategy to mitigate fibrosis in this challenging disease. Further research is underway to fully explore the therapeutic potential and genes and pathways involved in RNA methylation and its impact on fibrotic diseases such as SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/surveying-rna-methylation-in-scleroderma-highlights-roles-for-demethylases-alkbh5-and-fto-in-fibrosis/