Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes

Abstract

Background: As the application of carbon nanotubes (CNT) in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT) could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans.

Methods: In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8), were compared with expression profiles of non-exposed (n = 7) workers (e.g., professional and/or technical staff) from the same manufacturing facility.

Results: Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs.

Conclusion: This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers of MWCNT exposures in humans.

Fig 1. Transmission electron micrographs of samples collected in MWCNT facilities.

TEM images of filters collected from personal breathing zone sampling showing: A, more dispersed MWCNT particles; B-C, agglomerated structures from potentially exposed groups, and D, a media blank (stored in a clean room).

Fig 2. An overall schema summarizing the integrated bioinformatics analysis of differentially expressed ncRNAs and mRNAs to determine their potential relationships and interactions in MWCNT exposed and non-exposed workers.

Fig 3. Highest scoring gene molecular networks of mRNAs and miRNA-mRNA target pairs in MWCNT high exposure group.

Gene signaling network analysis by IPA of differentially expressed (A) mRNAs and (B) miRNA-mRNA target pairs in high MWCNT exposure group. The datasets either containing all differentially expressed genes or genes having consistent expression direction based on differentially expressed miRNAs were uploaded into IPA then analyzed with the cutoff criteria of ≥ 1.5 fold change and a p-value < 0.05. Only the network is presented for each group. Intensity of the red (upregulated) or green (down regulated) color of the nodes in the graph indicates level of gene expression.

Fig 4. KEGG pathway enrichment analysis of dysregulated genes and miRNA-mRNA target pairs in the MWCNT-exposed workers.

Venn diagrams showing common and unique enriched pathways of (A) up- and down-regulated mRNAs KEGG pathways, and (D) down-regulated mRNAs and miRNA-mRNAs targets identified using micro-TDS and TargetScan algorithms of DIANA-miRPath.

Fig 5. The regulatory network of ncRNAs and mRNAs in workers exposed to MWCNTs.

The regulatory networks of significantly dysregulated lncRNAs and mRNAs (p-value<0.05, fold-change>±1.5) having positional and/or functional relationship with (A) up-regulated miRNAs (p<0.05, fold-change>1.5) and (B) down-regulated miRNAs (p>0.05, fold-change>-1.5), generated using IPA. The lncRNAs, represented using red (↓) and green (↑) rectangles and dotted lines, with consistent co-expression as mRNAs and/or containing miRNA target binding sites from starBase v2.0 were manually added to the network.