Altered long non-coding RNAs expression in normal and diseased primary human airway epithelial cells exposed to diesel exhaust particles

Abstract

Background: Exposure to diesel exhaust particles (DEP) has been linked to a variety of adverse health effects, including increased morbidity and mortality from cardiovascular diseases, chronic obstructive pulmonary disease (COPD), metabolic syndrome, and lung cancer. The epigenetic changes caused by air pollution have been associated with increased health risks. However, the exact molecular mechanisms underlying the lncRNA-mediated pathogenesis induced by DEP exposure have not been revealed.

Methods: Through RNA-sequencing and integrative analysis of both mRNA and lncRNA profiles, this study investigated the role of lncRNAs in altered gene expression in healthy and diseased human primary epithelial cells (NHBE and DHBE-COPD) exposed to DEP at a dose of 30 μg/cm².

Results: We identified 503 and 563 differentially expressed (DE) mRNAs and a total of 10 and 14 DE lncRNAs in NHBE and DHBE-COPD cells exposed to DEP, respectively. In both NHBE and DHBE-COPD cells, enriched cancer-related pathways were identified at mRNA level, and 3 common lncRNAs OLMALINC, AC069234.2, and LINC00665 were found to be associated with cancer initiation and progression. In addition, we identified two cis-acting (TMEM51-AS1 and TTN-AS1) and several trans-acting lncRNAs (e.g. LINC01278, SNHG29, AC006064.4, TMEM51-AS1) only differentially expressed in COPD cells, which could potentially play a role in carcinogenesis and determine their susceptibility to DEP exposure.

Conclusions: Overall, our work highlights the potential importance of lncRNAs in regulating DEP-induced gene expression changes associated with carcinogenesis, and individuals suffering from COPD are likely to be more vulnerable to these environmental triggers.

Keywords: COPD; Diesel exhaust particle; carcinogenesis; lncRNA-mRNA interaction; lncRNAs; susceptibility.

Figure 1:

A schematic diagram for the timeline of cell culture, ALI differentiation, and the subsequent DEP exposure.

Figure 2:. Cytotoxicity in NHBE and DHBE-COPD cells following DEP exposure.

Cells were exposed to DEP at concentrations of 10 μg/cm², 20 μg/cm², and 30 μg/cm² for 24 h. LDH release was used to calculate the percentage of cytotoxicity compared to negative controls of unexposed cells maintained in cell media and positive controls treated with Triton X-100 (0.1% v/v). To determine statistical significance in comparison to the negative controls, two-way ANOVA was used; ns: not significant (p > 0.05), *** (p < 0.001), and **** (p < 0.0001).

Figure 3:. Differential expression of mRNAs (DE mRNAs) in NHBE and DHBE-COPD cells.

DE mRNA volcano plots in (a) NHBE and (b) DHBE-COPD cells after DEP exposure. The X-axis represents the log 2-fold change, while the Y-axis represents the adjusted p values: −log 10 (padj). The blue dots represent significantly upregulated mRNAs, while red dots represent significantly downregulated mRNAs. Non-differentially expressed mRNAs are represented by black dots. (c) A Venn diagram depicts the number of distinct and common (overlapping) DE mRNAs in NHBE and DHBE-COPD cells.

Figure 4:. Differential expression of lncRNAs (DE lncRNAs) in NHBE and DHBE-COPD cells.

DE lncRNA volcano plots of in (a) NHBE and (b) DHBE-COPD cells after DEP exposure. The X-axis represents log 2-fold change, while the Y-axis represents the adjusted p values: −log 10 (padj). Blue dots represent significantly upregulated lncRNAs, while red dots represent significantly downregulated lncRNAs. Non-differentially expressed lncRNAs are represented by black dots. (c) A Venn diagram shows the number of distinct and common (overlapping) DE lncRNAs in NHBE and DHBE-COPD cells.

Figure 5:. Predicted trans-targeted genes (log2FC>|±1|) and regulatory network of DE lncRNAs in NHBE cells.

The DE lncRNAs regulatory network was built for the NHBE cells using the R package (version 3.6.3). The colors represent the different types of RNAs; orange: mRNA, and green: lncRNA. The triangles represent upregulation, while the dots represent downregulation.

Figure 6:. Predicted trans-targeted genes (log2FC>|±1|) and regulatory network of DE lncRNAs in DHBE-COPD cells.

The DE lncRNA regulatory network was constructed for DHBE-COPD cells using the R package (version 3.6.3). The colors represent the different types of RNAs; blue: mRNA, and red: lncRNA. The triangles represent up-regulation, while the dots represent downregulation.

Figure 7:

A schematic diagram of DEP exposure-mediated health outcomes in the NHBE and DHBE-COPD cells.