Transcriptomic Analyses of the Biological Effects of Airborne PM2.5 Exposure on Human Bronchial Epithelial Cells

<div><p>Epidemiological studies have associated high levels of airborne particulate matter (PM) with increased respiratory diseases. In order to investigate the mechanisms of air pollution-induced lung toxicity in humans, human bronchial epithelial cells (16HBE) were exposed to various concentrations of particles smaller than 2.5 μm (PM2.5) collected from Beijing, China. After observing that PM2.5 decreased cell viability in a dose-dependent manner, we first used Illumina RNA-seq to identify genes and pathways that may contribute to PM2.5-induced toxicity to 16HBE cells. A total of 539 genes, 283 up-regulated and 256 down-regulated, were identified to be significantly differentially expressed after exposure to 25 μg/cm² PM2.5. PM2.5 induced a large number of genes involved in responses to xenobtiotic stimuli, metabolic response, and inflammatory and immune response pathways such as MAPK signaling and cytokine-cytokine receptor interaction, which might contribute to PM2.5-related pulmonary diseases. We then confirmed our RNA-seq results by qPCR and by analysis of IL-6, CYP1A1, and IL-8 protein expression. Finally, ELISA assay demonstrated a significant association between exposure to PM2.5 and secretion of IL-6. This research provides a new insight into the mechanisms underlying PM2.5-induced respiratory diseases in Beijing.</p></div

Sequences of primers used in quantitative RT-PCR.

<p>Sequences of primers used in quantitative RT-PCR.</p

The top 10 biological processes identified by GO analysis of differentially expressed genes.

<p>Bars represent-log(P-value). P-value was calculated by hypergeometric test.</p

Top 10 up-regulated genes in PM2.5 treated 16HBE cells.

<p>Top 10 up-regulated genes in PM2.5 treated 16HBE cells.</p

Analysis of differentially expressed genes between PM2.5 treated and untreated samples.

<p>A. Gene expression level of control versus PM2.5 treated. FDR<0.001 and the absolute value of log2 fold change ≥ 1 were used as the threshold to judge the significance of gene expression difference. B. Summary of the numbers of differentially expressed genes in the PM2.5 treated sample versus untreated control.</p

The significant pathways for the DEGs.

<p>The arrows ↑ and ↓ indicate up- or down-regulation.</p

Top 10 down-regulated genes in PM2.5 treated 16HBE cells.

<p>Top 10 down-regulated genes in PM2.5 treated 16HBE cells.</p

Correlation between RNA-seq data and qRT-PCR data.

<p>The fold changes were calculated using the 2^-△△Ct method comparing PM2.5 treated cell to control untreated samples. All Ct values were normalized to GAPDH.</p

The fold changes in CYP1A1, IL-8 and IL-6 expression between control and PM2.5 treated 16HBE cells confirmed by western blot analysis.

<p>A-C. The expression of CYP1A1, IL-8 and IL-6, respectively, detected by western blot analysis of cell lysates. In each gel image the left lane (1) contained untreated control cells and the right lane (2) contained cells treated with 25 μg/cm² PM2.5. GAPDH was used as a loading control. D. Quantitation of western blot band intensity (n = 3). Data is expressed as mean ± SD. (*) indicates significant difference (p < 0.05) when compared to control, (**) represents significant difference (p < 0.01) compared to control.</p

The effect of PM2.5 concentrations on cell viability in 16HBE cells.

<p>Cells were exposed to various concentrations of PM2.5 (1.56, 7.81, 15.63, 31.25 and 62.50 μg/cm² (5, 25, 50, 100 and 200 μg/mL) for 24 hours. Cell viability was expressed as a percentage of unexposed control (mean ± S.D.). (*) indicates significant difference (p < 0.05) when compared to control, (**) represents significant difference (p < 0.01) compared to control.</p