Raveen Badyal https://orcid.org/0000-0002-1278-3933 Beth A. Whalen Gurpreet K. Singhera Basak Sahin Kevin J. Keen Christopher J. Ryerson Pearce Wilcox James V. Dunne


Introduction: Scleroderma (SSc) is an autoimmune disorder with the hallmark of fibrosis of the skin, vasculature and internal organs. Patients with SSc and undifferentiated connective tissue disease (UCTD) are susceptible to interstitial lung disease (ILD), leading to decreased lung function and death. Idiopathic pulmonary fibrosis (IPF) is a form of ILD that is not associated with extrapulmonary manifestations. In this study, lung involvement of SSc was studied by observing how disease progression and pathogenesis differ among patients with SSc, UCTD, and ILD compared to healthy controls and patients with IPF. Our group has previously identified disease targets through microRNA sequencing, including the DICER enzyme, which works closely with the protein DGCR8 and the enzyme DROSHA in the RNA interference pathway. The canonical pathway stipulates that DICER processes microRNAs in the cytosol while DGCR8 and DROSHA process microRNAs in the nucleus. DICER, DROSHA, and DGCR8 are hypothesized to contribute to ILD progression.
Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from voluntary participants, including healthy controls. PBMCs were subsequently lysed with subcellular fractionation buffer. Western blotting was done on the resulting cytosolic and nucleic fractions for DICER, DROSHA, and DGCR8 protein expression. The cytosolic fractions were normalized to GAPDH, while the nucleic fractions were normalized to B2M. Nonparametric Kruskal‐Wallis tests were used for statistical analysis.
Results: The medians were significantly higher for healthy controls for DICER in the nucleus with a p-value of 0.0302, and DROSHA in the cytosol with a p-value of 0.0406 compared to patients with SSc, UCTD, and IPF.
Discussion: Differences in expression were found for DROSHA in the cytosol and DICER in the nucleus, suggesting dysregulation of the non-canonical RNA interference pathways in SSc, UCTD, and IPF patients. Variability of disease progression within the groups could lead to variable enzyme and protein levels within the same disease status. With larger sample sizes, statistically insignificant differences would become significant. Lipid nanoparticle technology could be used to deliver deficient microRNAs to silence mRNA in patients.
Conclusion: Due to dysregulation of the RNA interference pathway, microRNAs may be inadequately processed in the patient groups.

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