RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.
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 micro...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3632591?pdf=render |
| _version_ | 1811214220004425728 |
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| author | B Alex Merrick Dhiral P Phadke Scott S Auerbach Deepak Mav Suzy M Stiegelmeyer Ruchir R Shah Raymond R Tice |
| author_facet | B Alex Merrick Dhiral P Phadke Scott S Auerbach Deepak Mav Suzy M Stiegelmeyer Ruchir R Shah Raymond R Tice |
| author_sort | B Alex Merrick |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T06:00:39Z |
| format | Article |
| id | doaj.art-a3b59a1659ee46fb996fa3a880f22e55 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T06:00:39Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| spelling | doaj.art-a3b59a1659ee46fb996fa3a880f22e552022-12-22T03:45:03ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0184e6176810.1371/journal.pone.0061768RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.B Alex MerrickDhiral P PhadkeScott S AuerbachDeepak MavSuzy M StiegelmeyerRuchir R ShahRaymond R TiceDeep 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.http://europepmc.org/articles/PMC3632591?pdf=render |
| spellingShingle | B Alex Merrick Dhiral P Phadke Scott S Auerbach Deepak Mav Suzy M Stiegelmeyer Ruchir R Shah Raymond R Tice RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. PLoS ONE |
| title | RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. |
| title_full | RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. |
| title_fullStr | RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. |
| title_full_unstemmed | RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. |
| title_short | RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. |
| title_sort | rna seq profiling reveals novel hepatic gene expression pattern in aflatoxin b1 treated rats |
| url | http://europepmc.org/articles/PMC3632591?pdf=render |
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