Salivary MicroRNA Signatures for Early Detection of Recurrent Aphthous Stomatitis

Abstract

Background: Recurrent aphthous stomatitis (RAS) is a common relapsing ulcerative disorder of the oral mucosa in which clinical assessment is largely subjective and lacks validated non-invasive biomarkers that reflect disease phase and recurrence burden. Salivary microRNAs (miRNAs) are stable biofluid analytes that may capture local inflammatory and epithelial injury–repair dynamics. Objective: To evaluate phase-specific salivary miRNA expression in RAS and determine associations with clinical severity and annual recurrence frequency. Methods: In an analytical cross-sectional observational study conducted over eight months at a tertiary care hospital in Lahore, Pakistan, 75 adults (18–45 years) were enrolled into three independent groups (n=25 each): active RAS (≤72 hours of ulcer onset), healing RAS (7–10 days post-onset), and healthy controls. Unstimulated whole saliva was collected under standardized conditions and analyzed by qRT-PCR for miR-21, miR-31, miR-146a, and miR-155, normalized to U6 and expressed using the 2^−ΔΔCt method. Pain (VAS), ulcer size (mm²), and annual recurrence frequency were recorded. Results: All miRNAs were significantly higher in active RAS than healing and controls (ANOVA p<0.001), with large phase-discrimination effects (η²=0.69–0.79). Active-phase expression peaked for miR-155 (3.45±0.57) and miR-21 (3.24±0.61). miRNA levels correlated strongly with pain and ulcer size (maximum r=0.81 and r=0.77 for miR-155; p<0.001) and with annual recurrence frequency (miR-155 r=0.74; miR-21 r=0.71; p<0.001). In multivariable regression adjusted for age and sex, miR-21 (β=0.42; p<0.001) and miR-155 (β=0.39; p<0.001) were independent predictors of recurrence (adjusted R²=0.64). Conclusion: Salivary miR-21 and miR-155 are robust phase-sensitive biomarkers in RAS and independently associate with recurrence burden, supporting their prioritization for non-invasive activity monitoring and recurrence stratification.