Chronic obstructive pulmonary disease (COPD) is a lung condition characterised by progressive airflow limitation in part due to narrowing and fibrosis of small airways. COPD is associated with cellular senescence which is driven by stressors such as oxidative stress. MicroRNA-21 (miR-21) and microRNA-34a (miR-34a) are upregulated in COPD, however their regulatory role in COPD pathogenesis or in response to oxidative stress remains unclear.
To better understand the role of miR-21 and miR-34a in COPD, a microfluidic platform was developed to quantify miR-21 and miR-34a molecules in single cells. Sandwich hybridisation assay was optimised and integrated into microfluidic chambers for single cell miRNA detection. The sensitivity was demonstrated by quantifying levels of miRNA in nasal cells and fluid. Levels of miR-21 were varied in nasal cells and fluid within and between individuals.
Levels of miR-21 and miR-34a were increased in small airway epithelial cells (SAEC) and fibroblasts (SAF) from COPD subjects compared to non-smokers, and were varied within and between subjects. MiR-21 and miR-34a were detected simultaneously from the same cell using a multiplex assay which showed a positive correlation between miR-21 and miR-34a expressed in SAEC and SAF.
Baseline gene expression of miR-21 and miR-34a targets differed in SAEC and SAF. Altered levels of miR-21 and miR-34a influenced their targets mRNA and protein levels, however the effect was different in COPD cells compared to healthy cells. MiR-21 and miR-34a levels were elevated in response to oxidative stress, while their target gene expression was reduced and senescent markers were increased.
This study demonstrated that a microfluidic platform can be developed to quantify single miRNA molecules in single cells to determine cell-to-cell variation of miRNAs within cell populations. MiR-21 and miR-34a are crucial regulators in COPD and may have a protective role in response to oxidative stress, however further investigation is required.