Polycyclic aromatic hydrocarbons (PAHs) are a class of contaminants ubiquitous in the environment and result from the incomplete combustion of fossil fuels. Many PAHs have been identified as procarcinogenic, and are metabolized to form DNA adducts; however, other mechanisms also may contribute to toxicity and help explain differences in toxicity across the wide class of compounds. This study examines the role of microRNAs (miRNAs) in mediating toxicity by benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC) in 3D human bronchial epithelial cells (HBEC). miRNAs are short (≤22 nt), non-coding RNA molecules that post-transcriptionally regulate gene expression. Recent studies have identified dysregulation of miRNA expression in response to BAP, cigarette smoke, and pollution-related lung diseases and cancers.
The 3D HBEC model provides a more accurate representation of the effects of PAH toxicity because it is metabolically competent, containing multiple cell types in a pseudostratified model that is more representative of in vivo exposure. This study aims to understand the functional consequences of miRNAs in HBEC after exposure to BAP and DBC, which have previously been found to function through unique mechanisms. Cells were exposed to 500 ug/ml BAP and 10 ug/ml DBC for 48 hrs and samples were collected for RNA isolation and parallel analysis of mRNA and miRNA by RNA sequencing using Illumina HiSeq 3000.
Significant (q<0.05) differentially expressed miRNA and mRNA were analyzed in an anti-correlated fashion in Bioinformatics Resource Manager to identify miRNA-mRNA interactions and visualized as networks in Cytoscape to identify patterns of regulation reflecting a response to PAHs. DBC treatments showed more regulation of unique miRNA, with 53 significantly down- and 46 up-regulated miRNAs, compared to BAP’s 14 down- and 35 up-regulated miRNAs. These miRNAs targeted 546 up- and 654 down-regulated genes significant in the DBC dataset, and 176 up- and 750 down-regulated in the BAP. miRNAs uniquely up-regulated in BaP were linked with a more significant response in cell adhesion and developmental processes. In DBC, up-regulated miRNAs showed more significant response overall in regulation of cell cycle, translation, and apoptosis. Processes perturbed by down-regulated miRNA were less consistent, with BaP showing more effect for cytoskeletal and cell cycle processes, while DBC shows more response for cell adhesion and DNA damage. These data are the first to describe the role of miRNAs as regulators of PAH toxicity in primary human 3D HBEC and could represent important mechanisms associated with PAH-mediated lung disease and cancer in humans.