Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.
PLoS One. 2013 Apr 22;8(4):e61768. doi: 10.1371/journal.pone.0061768. Print 2013.
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.
使用深度测序技术研究了 1ppm 黄曲霉毒素 B1（AFB1）对雄性大鼠肝脏转录组的亚慢性影响，在组织病理学病变或肿瘤发生之前。我们假设 RNA-Seq 会比微阵列分析揭示更多差异表达基因（DEG），包括与 AFB1 致癌活性相关的低拷贝和新转录本，与饲料对照（CTRL）相比。使用 TopHat 将配对末端读数映射到大鼠基因组（Rn4），并进一步通过 DESeq 和 Cufflinks-Cuffdiff 分析来识别差异表达的转录本、新外显子和未注释的转录本。DEG 的 PCA 和聚类分析表明，AFB1 和 CTRL 处理之间有明显的分离，并且组重复之间有一致性。对 8 个高和中 DEG 和 3 个低 DEG 的 qPCR 显示 RNA-Seq 和微阵列转录本之间具有良好的可比性。DESeq 分析鉴定出 1026 个差异表达转录本，与微阵列相比，差异倍数大于两倍（p<0.005），这是由于 RNA-Seq 转录本的碱基对分辨率、探针在转录本中的放置或缺乏探针来检测新的转录本、剪接变体和外显子。DEG 中的途径分析表明，Ahr、Nrf2、GSH、外源性物质、细胞周期、细胞外基质和细胞分化途径的信号传导与导致 AFB1 致癌的途径一致，包括近 200 个上调的转录本，这些转录本受到与动粒结构、有丝分裂纺锤体组装和组织重塑相关的 E2f1 相关途径的控制。我们报告了 49 个新的差异表达转录本，包括通过 PCR-克隆两个独特的、未注释的、肝脏 AFB1 反应性转录本（HAfT）在染色体 1.q55 和 15.q11 上的确认，这两个转录本的表达上调了 10 到 25 倍。发现了几个潜在的新外显子，并对外显子进行了细化，包括 AFB1 外显子特异性诱导同源家族成员 Ugt1a6 和 Ugt1a7c。我们发现大鼠转录组中包含许多以前未识别的、AFB1 反应性外显子和转录本，这支持 RNA-Seq 提供对 AFB1 介导的基因表达导致肝细胞癌的新见解的能力。
