Profiling Non-Coding RNA Changes Associated with 16 Different Engineered Nanomaterials in a Mouse Airway Exposure Model

Abstract

Perturbations in cellular molecular events and their associated biological processes provide opportunities for hazard assessment based on toxicogenomic profiling. Long non-coding RNAs (lncRNAs) are transcribed from DNA but are typically not translated into full-length proteins. Via epigenetic regulation, they play important roles in organismal response to environmental stress. The effects of nanoparticles on this important part of the epigenome are understudied. In this study, we investigated changes in lncRNA associated with hazardous inhalatory exposure of mice to 16 engineered nanomaterials (ENM)-4 ENM (copper oxide, multi-walled carbon nanotubes, spherical titanium dioxide, and rod-like titanium dioxide particles) with 4 different surface chemistries (pristine, COOH, NH₂, and PEG). Mice were exposed to 10 µg of ENM by oropharyngeal aspiration for 4 consecutive days, followed by cytological analyses and transcriptomic characterization of whole lung tissues. The number of significantly altered non-coding RNA transcripts, suggestive of their degrees of toxicity, was different for each ENM type. Particle surface chemistry and shape also had varying effects on lncRNA expression. NH₂ and PEG caused the strongest and weakest responses, respectively. Via correlational analyses to mRNA expression from the same samples, we could deduce that significantly altered lncRNAs are potential regulators of genes involved in mitotic cell division and DNA damage response. This study sheds more light on epigenetic mechanisms of ENM toxicity and also emphasizes the importance of the lncRNA superfamily as toxicogenomic markers of adverse ENM exposure.

Keywords: long non-coding RNA; nanoparticles; toxicogenomics.

Figure 1

Comparison of most biologically relevant differentially expressed (DE) lncRNA transcripts across the different materials and surface chemistry types. In (A), the number of DE transcripts is shown for all engineered nanomaterials, across all the different material surface chemistry groups. Venn comparisons reveal both unique and shared lncRNA expression signatures between ENM (B) and ENM surface chemistries (C).

Figure 2

Heatmap of differentially expressed lncRNA transcripts (1.5-fold change, q-value 0.05). Hierarchical clustering reveals 3 distinct clusters of lncRNAs. Boxes highlight a group of lncRNA transcripts that are upregulated in each cluster (I–III). Each column represents the average Z-scored intensity value from 3 biological replicates.

Figure 3

Co-regulated lncRNA-mRNA networks, triggered by hazardous copper oxide (CuO), multiwalled carbon nanotubes (MWCNT) and titanium dioxide (TiO₂p/TiO₂r) engineered nanomaterial exposures. Hierarchical clustering based on Pearson’s correlation coefficients between differentially expressed lncRNA and mRNA transcripts, reveals 5 clusters of lncRNA transcripts that are highly correlated (−0.8 < R > 0.8) to at least 15 genes (mRNA) (A). The positively correlated mRNA-lncRNA transcript pairs from the top two correlated clusters; lncRNA (cluster #1 & #3) and mRNA (cluster #1 & #3) are shown in (B). No differentially expressed lncRNA transcripts from the TiO2r exposures are correlated (−0.8 < R > 0.8) to differentially expressed genes. Distribution of lncRNA-associated genes between the CuO, MWCNT, and TiO₂p exposures are shown in (C). Shared and unique lncRNA-associated differentially expressed genes indicate both common and unique epigenetic mechanisms of toxicity.

Figure 4

Pathway enrichment analysis of genes identified in lncRNA-mRNA co-expression networks. Two main clusters of mRNA were found to be associated (Pearson’s correlation coefficient, p-value < 0.05, −0.8 < R > 0.8) to lncRNA expression. The genes in cluster #1, left panel, were predominantly involved in biological processes related to cellular response to chemical, xenobiotics, or drug stimulus. Cluster #3, with 4 times more genes (right panel), was highly enriched for genes that control the cell cycle, chromosomal organization, and DNA damage/repair response.

Figure 5

Categorization of ENM toxicity based on expression of lncRNAs involved in regulation of DNA damage and repair response. In (A), the frequency distribution of DE lncRNA transcripts across the various engineered nanomaterial (ENM) exposures is shown. The highest number of these regulatory lncRNAs were identified as differentially expressed in CuO exposures, with the exception of CuO-PEG (red font), followed by all MWCNT plus TiO₂p-NH₂/PEG (black font). A few to none of these lncRNAs were differentially expressed in all TiO₂r exposures, CuO-PEG, and TiO₂p-Core/COOH (gray font). A heatmap of the hierarchical clustering that is based on the average relative (Z-score) expression of these lncRNAs across all sample groups is shown in (B). Clusters of the sample groups that ranks the relative toxicity of the ENM in very much the same way as seen in (A). That is, from zero (unexposed)/low (gray box) to mid toxicity (black box) and then high toxicity (red box) ENM.