RNA-Seq Profiling Reveals Novel Hepatic Gene Expression Pattern in Aflatoxin B1 Treated Rats

Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.
PLoS ONE (Impact Factor: 3.23). 04/2013; 8(4):e61768. DOI: 10.1371/journal.pone.0061768
Source: PubMed


Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq's capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma.

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Available from: Raymond R Tice
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    • "System-wide studies have been changing as new techniques become available; sequencing has changed the way genome-wide toxicology data is generated. Where microarrays have previously been used to generate expression profiles , RNA collection followed by deep sequencing (RNAseq ) has enhanced several toxicological studies [41] [42] [43]. For example, a recent study by Yang and colleagues (2014) used RNA-seq to elucidate the molecular effects of crotonaldehyde exposure on macrophage-like cells [44]. "
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    • "Cufflinks [8], [18], [23], DESeq [6], edgeR [22], etc.). Taking advantage of these tools, the power of the RNA-Seq approach to detect DEGs has been recently demonstrated [24]–[27]. However, there is no consensus on which analysis method is optimal, nor on how to approach a proper analysis to ensure validity of outcomes in terms of reproducibility, accuracy and robustness. "
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