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

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. The objective of the study was 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 patients with COPD. Small airway epithelial cells (SAEC) and fibroblasts (SAF) were isolated from patients with COPD and nonsmokers (<i>n</i> = 6-8 per group). A microfluidic chip with dual miRNA sandwich hybridization 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. Single-cell analysis revealed significantly higher miR-21-5p and miR-34a-5p expression in COPD-derived cells compared with 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. 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.<b>NEW & NOTEWORTHY</b> This study demonstrates a novel microfluidic system that can precisely quantify two specific microRNAs in COPD samples at a single-cell resolution using small quantities of cells and reveal the cellular heterogeneity in cell populations. This dual detection system enables the interrelationship between microRNAs in the same cell to be studied as well as having the ability to monitor microRNA levels during disease progression and therapeutics.