Introduction

COPD is associated with airflow limitation as a consequence of the combined effects of emphysema, airway wall thickening, mucus obstruction, and peribronchiolar fibrosis which presents clinically as cough, sputum production, and progressive dyspnea. The severity of these abnormalities varies between patients but a subset is particularly impacted by mucus dysfunction including hypersecretion and impaired mucociliary clearance which can lead to mucus plugging. The mechanisms underlying this mucus dysfunction are complex and incompletely understood though exposure to cigarette smoke (CS), aeroallergens, and particulate and gas pollution can contribute.

Cadmium (Cd) is a toxicant that humans are exposed to through air, food and water. Combustion releases cadmium oxide which can be easily adsorbed on the particulate matter (PM) of < 2.5 including diesel exhaust particles or CS. CS which has approximately 2–3 µg of Cd per cigarette is associated with an increased number of mucus producing cells, impaired mucus clearance and increased mucosal permeability to allergens1. But the mechanisms contributing to this mucosal dysfunction have many facets.

The evolutionarily conserved small non-coding RNAs (miRNAs) have been studied to determine their role in the regulation of protein translation and their contribution to disease pathophysiology2. miRNA or lncRNAs may be positively or negatively regulated by epigenetic regulators like EZH2. Heavy metals are not only known to induce changes in protein expression profiles but also have an effect on microRNA through epigenetic marker regulation3. Prediction of Cd-responsive miRNAs suggested regulation of 214 target genes4. ANO1 is a mechano-sensitive chloride channel, preferentially Ca2+ activated, encoded by the ANO1 gene located on human chromosome 11q13. A 960 amino acid protein with 10 membrane spanning segments it is expressed on the apical region of a variety of epithelial cells such as gastrointestinal, bronchial and pulmonary vessels smooth muscle cells and endothelial cells of arteries. Studies have reported regulation of ANO1 by miR-9, miR-144, miR-181a-2-3p and miR-3815,6,7,8. miR-9 in bronchial epithelial cells negatively regulates ANO1 expression in cystic fibrosis (CF)9. In a study of CF subjects, expression of miR-9 is higher which suppresses the expression of ANO1 by directly binding to the 3ʹ UTR of ANO1 mRNA. This study proposed miR-9 as a potential target for anti-CF therapy where its downregulation increases ANO1 associated chloride efflux to potentiate mucociliary clearance5. Also, knockout of ANO1 was shown to reduce Cl− conductance and inhibit mucus secretion. Interestingly, activation of ANO1 by denufosol induced cough and failed to provide any benefit to CF patients10. In chronic lung diseases such as asthma and CF which have high susceptibly to environmental pollutants/allergens expression of ANO1 in airway epithelial cells is upregulated in the presence of mucus expression inducing interleukins- IL-4, IL-8 and IL-135,11,12. In asthma increased ANO1 is shown to effectively modulate MUC5AC expression and mucus production. However, in COPD expression of ANO1 is not well studied. Since ANO1 supports fluid secretion and airway smooth muscle contraction, inhibition rather than activation is suggested as an appropriate treatment mechanism for inflammatory airway diseases. miR-381 was our target miRNA for this study because its association is closely related to barrier dysfunction and cell proliferation and ANO1 is most recognized for cellular functions such as cell proliferation, attachment and a controversial role in the secretion of mucus5,13,4c).

TGF-β signaling which has been extensively studied and reported to highly expressed and promote airway remodeling in asthma is also suppressed by an increased expression of miR-381. TGF-β promotes goblet cell transformation resulting in increased mucus production and secretion28. Knockdown of miR-381 increases epithelial cell proliferation with increased Ki-67 stained cells and intensity13. In gastric cancer induction of miR-381 expression suppresses differentiation of epithelial cells and reduces cell proliferation29. Our observations from experiments involving low-dose Cd-exposure of epithelial cells suggested a similar understanding that ANO1 is a direct target for miR-381 which is downregulated upon low-dose Cd-exposure (Fig. 4c). Thus, CS-induced Cd-toxicity may alter cellular homeostasis mechanisms at very low concentrations. Thus Cd-exposure in a person with an existing pulmonary condition can have an additive or adverse effect with increased susceptibility towards infections and environmental allergens and that miRNAs may act as potential therapeutic targets to be explored further in Cd-exposure and subsequent lung injury.