Digital PCR-Based Assessment of Piwi-RNA PIR35982 as a Supporting Biomarker in Rheumatoid Arthritis

Marek Cieśla,1 Dorota Darmochwał-Kolarz,2 Hubert Kubis,1 Bogdan Kolarz3 1Laboratory of Diagnostic and Clinical Epigenetics, Faculty of Medicine, University of Rzeszow, Rzeszow, Poland; 2Department of Obstetrics and Gynecology, Faculty of Medicine, University of Rzeszow, Rzeszow, Poland; 3Department of Rheumatology, Faculty of Medicine, University of Rzeszow, Rzeszow, PolandCorrespondence: Marek Cieśla, Laboratory of Diagnostic and Clinical Epigenetics, Faculty of Medicine, University of Rzeszow, Kopisto 2a St, Rzeszow, 35-959, Poland, Tel +48 178516851, Email [email protected]: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder with an etiology that remains incompletely understood. Its development is influenced by both environmental and molecular factors, with growing attention to epigenetic mechanisms. Among these, PIWI-interacting RNAs (PIRs) have emerged as molecules of interest. PIRs are small, single-stranded noncoding RNAs, and play a crucial role in maintaining genome stability in germline cells; however, they were also found in somatic cells.Purpose: The aim of this study was to identify PIRs whose expression may be associated with the severity of RA.Patients and Methods: A total of 57 individuals were enrolled in the study, including 37 patients with RA and 20Â healthy controls (HCs). Quantitative real-time polymerase chain reaction (qPCR) and digital polymerase chain reaction (dPCR) were used to evaluate the plasma concentrations and peripheral blood mononuclear cells (PBMCs) expression levels of selected PIRs.Results: In PBMCs, PIR27124 showed elevated expression in RA compared to HCs (p=0.04). Additionally, both PIR27731 and PIR35982 showed increased expression in rheumatoid factor (RF)-negative RA patients relative to RF-positive patients (p-values 0.045 for both). In plasma, PIR35982 showed a decreased concentration in RA compared to HCs (p=0.001). Furthermore, both qPCR and dPCR confirmed that RF-negative RA patients had decreased plasma levels of PIR35982 compared to HCs (p=0.01 and p=0.02, respectively).Conclusion: PIR35982 can be considered a potential molecular marker associated with RA and may be useful for identifying RF-negative patients. Moreover, dPCR represents a promising tool for the discovery and evaluation of novel biomarkers, such as PIRs.Keywords: digital PCR, epigenetic, molecular marker, piwi-RNA, rheumatoid arthritis