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MicroRNA-mediated changes contributing to susceptibility for benzo[a]pyrene toxicity in a 3D respiratory model for asthma

ABSTRACT: There is increased emphasis on understanding how non-chemical stressors that contribute to inflammation in the lung may influence adverse health outcomes after chemical exposures. Prior studies in an in vitro respiratory model of type 2 asthmatic inflammation found cells from the asthmatic phenotype respond uniquely to benzo[a]pyrene (BAP) treatment compared to normal cells across multiple endpoints related to mucus production, goblet cell hyperplasia, mucociliary dysfunction and airway remodeling. To further understand how cellular response to BAP is regulated in a model of inflammation-based disease, this study examines changes in miRNA and mRNA regulation following BAP exposure in primary human bronchial epithelial cells (HBECs) cultured at the air-liquid interface with normal and interlukin-13 (IL-13) induced asthmatic phenotypes. Primary 3D HBECs differentiated in the presence and absence of 10ng/mL IL-13 were treated on day 25 with 158 uM BAP for 48 hours. Differentially expressed (q<0.01) miRNA and mRNA were analyzed to predict miRNA target interactions and assess the functional consequences of miRNAs in each phenotype. While BAP-treated HBEC with the IL-13 asthmatic phenotype had a similar number of differentially expressed miRNA (93 up- and 100 down-regulated) compared to BAP-treated normal HBEC (93 up- and 94 down-regulated), IL-13 HBEC treated with BAP were shown to have unique overrepresentation of miRNA targets involved in up-regulation of cell cycle processes and down-regulation of processes related to NOTCH, WNT, and Hedgehog signaling. These data are the first to provide insight into the role of miRNAs as regulators of chemical toxicity in a respiratory model of inflammation-based disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE305614 | GEO | 2025/12/01

REPOSITORIES: GEO

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Project description:There is increased emphasis on understanding cumulative risk from the combined effects of chemical and non-chemical stressors as it relates to public health. Recent animal studies have identified pulmonary inflammation as a possible modifier and risk factor for chemical toxicity in the lung after exposure to inhaled pollutants; however, little is known about specific interactions and potential mechanisms of action. In this study, primary human bronchial epithelial cells (HBEC) cultured in 3D at the air-liquid interface (ALI) are utilized as a physiologically relevant model to evaluate the effects of inflammation on toxicity of polycyclic aromatic hydrocarbons (PAHs), a class of contaminants generated from incomplete combustion of fossil fuels. Normal HBEC were differentiated in the presence of IL-13 for 14 days to induce a profibrotic phenotype similar to asthma. Fully differentiated normal and asthmatic phenotype HBEC were treated with benzo[a]pyrene (BAP; 1 – 40 ug/mL) or 1% DMSO/PBS vehicle at the ALI for 48 hrs. Cells were evaluated for cytotoxicity, barrier integrity, and transcriptional biomarkers of chemical metabolism and inflammation by quantitative PCR. Cells with the asthmatic phenotype treated with BAP show some significant (p<0.05) increase in cytotoxicity and significantly (p<0.05) decreased barrier integrity compared to normal cells. Asthmatic phenotype cells also showed increase sensitivity to BAP metabolism compared to cells with the normal phenotype. Additionally, RNA sequencing data showed that a large number of genes were uniquely significantly expressed in cells with the induced asthmatic phenotype exposed to BAP. Future studies will further explore mechanisms of toxicity from global transcriptomics and investigate the role of microRNA in mediating mechanisms of toxicity and inflammation. These data are the first to evaluate the role of combined environmental factors associated with inflammation from pre-existing disease and PAH exposure on pulmonary toxicity in a physiologically relevant human in vitro model.

Project description:We have developed cdk4/hTERT-immortalized normal human bronchial epithelial cells (HBECs) to study lung cancer pathogenesis. By studying the oncogenic effect of common lung cancer alterations (p53, KRAS, and c-MYC) we demonstrate the ability of this model to characterize the stepwise transformation of bronchial epithelial cells to full malignancy. Using HBECs derived from multiple individuals we found: 1) the combination of five genetic alterations (p53, KRASV12, c-MYC, CDK4 and hTERT) is sufficient for full tumorigenic conversion of HBECs; 2) high levels of KRASV12 are required for full malignant transformation of HBECs, however these levels also stimulate oncogene-induced senescence; 3) RAS-induced senescence is largely bypassed with loss of p53 function; 4) over-expression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; 5) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; 6) serum-induced epithelial-to-mesenchymal transition (EMT) increases in vivo tumorigenicity; 7) genetically-identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation as well as sensitivity to standard platinum-based chemotherapies; and 8) an mRNA signature derived from tumorigenic and non-tumorigenic clones is predictive of outcome in lung cancer patients. Collectively, we demonstrate this HBEC model system can be used to study the effect of oncogenic mutations on malignant progression, oncogene-induced senescence, and EMT along with clinically translatable applications such as development of prognostic signatures and drug response phenotypes. Human bronchial epithelial cells (HBECs) immortalized with cdk4 and hTERT were transformed with p53 knockdown, KrasV12 and cMYC over-expression and profiled on Illumina HumanHT-12 V4.0 expression beadchips. Transformed HBECs were grown in two different growth media: KSFM (defined, serum-free medium) or R10 (RPMI with 10% FBS) as indicated. Clones were isolated from HBECs with sh-p53 + KrasV12 and sh-p53 + KrasV12 + cMYC.

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MicroRNA-mediated changes contributing to susceptibility for benzo[a]pyrene toxicity in a 3D respiratory model for asthma

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