Single cell microfluidic quantification of miRNA-21 and miRNA-34a reveals miRNA interactions in small airway epithelial cells and fibroblasts from COPD patients

Rationale: MicroRNA-21 and microRNA-34a are implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but their cell-specific expression patterns and interactions within individual airway cells remain unexplored. Objective: To develop a single cell microfluidic platform for dual, amplification-free detection of miR-21-5p and miR-34a-5p in primary small airway cells from COPD patients. Methods: Small airway epithelial cells (SAEC) and fibroblasts (SAF) were isolated from COPD patients and non-smokers (n = 6-8 per group). A microfluidic chip with dual miRNA sandwich hybridisation assays was used to quantify miR-21-5p and miR-34a-5p in single cells. Expression of miRNAs and their target genes was evaluated under oxidative stress using qPCR and Western blotting. Main Results: Single cell analysis revealed significantly higher miR-21-5p and miR-34a-5p expression in COPD cells compared to controls. MiR-21 exhibited greater variability than miR-34a, and their positive correlation in control cells was disrupted in COPD. Oxidative stress elevated miR-21 and miR-34a while reducing expression of miR-21 targets and increasing senescence markers (p21 Cip1/Waf1 , p16 INK4a ). MiR-21 antagomir restored expression of suppressed targets in both cell types. Conclusions: Our novel single cell microfluidic platform enables precise, simultaneous detection of miR-21 and miR-34a in single small airway cells. This allows the interrelationship between the miRNAs to be assessed within the same cell. MiR-21 and miR-34a represent promising therapeutic targets for restoring gene regulatory balance in COPD.