Quantitative Analysis of Transcriptomes of Control and BP-3 exposure embryos (zebrafish) by RNA-sequencing

Purpose: The goals of this study are to quantitatively compare the expression difference of embryos incubated with or without benzophenone-3（BP-3） at the transcriptome level and find the underlying mechanism how could BP-3 impede the development of enteric nervous system. Methods: Intestinal RNA profiles of 5dpf Wide Type (WT) and BP-3 exposure groups of zebrafish embryos were generated by paired-end sequencing, in triplicate, using Illumina HiSeqTM 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level. The PossionDis algorithm was applied to perform differential gene detection and screened |log2 (FoldChange)| > 1 & qvalue<0.05 as the DEGs. Gene Set Enrichment Analysis (GSEA) comparing WT and BP-3 exposure groups was performed by GSEA software (version 4.0.03). A nominal p-value < 0.05 and false discovery rate (FDR) q-value < 0.25 were considered statistically significant for GSEA analyses. Results: The PossionDis algorithm was applied to perform differential gene detection and screened |log2 (FoldChange)| > 1 & qvalue<0.05 as the DEGs. RNA-seq data confirmed 159 up-regulated and 73 down-regulated genes in BP-3 exposure groups. Some MAPK/ERK signaling pathway related terms were enriched in molecular function and biological process and MAPK/ERK signaling pathway reached notable enrichment based on KEGG analysis. Conclusions: Our study represented the first detailed analysis of BP-3 exposure zebrafish intestinal transcriptomes, with biologic replicates, generated by RNA-sequencing technology. Our results showed that 159 genes were up-regulated and 73 were down-regulated in BP-3 exposure groups. Some MAPK/ERK signaling pathway related terms were enriched in molecular function and biological process. MAPK/ERK signaling pathway reached notable enrichment based on KEGG analysis. Intestinal RNA profiles of 5dpf Wide Type (WT) and BP-3 exposure groups of zebrafish embryos

研究目的：本研究旨在从转录组层面定量比较经二苯甲酮-3（benzophenone-3，BP-3）处理与未处理的胚胎的基因表达差异，探究BP-3阻碍肠神经系统发育的潜在分子机制。
方法：本研究以斑马鱼胚胎为实验材料，设置野生型（Wild Type, WT）组与BP-3暴露组，收集两组受精后5天（5 dpf）的肠道样本，采用Illumina HiSeq™ 2500平台完成三次生物学重复的双端测序，获取肠道转录组谱。对通过质量过滤的测序reads，在转录本异构体层面开展数据分析。采用PossionDis算法进行差异基因检测，以|log₂(FoldChange)|>1且q值<0.05作为差异表达基因（Differentially Expressed Genes, DEGs）的筛选标准。使用基因集富集分析（Gene Set Enrichment Analysis, GSEA）软件（版本4.0.03）对WT组与BP-3暴露组进行GSEA分析，以名义p值<0.05且错误发现率（False Discovery Rate, FDR）q值<0.25作为GSEA分析的统计学显著性阈值。
结果：本研究采用PossionDis算法完成差异基因检测，筛选得到符合阈值的DEGs。RNA测序（RNA-sequencing, RNA-seq）数据显示，BP-3暴露组中共鉴定出159个上调基因与73个下调基因。KEGG富集分析结果表明，丝裂原活化蛋白激酶/细胞外调节蛋白激酶（MAPK/ERK）信号通路相关条目在分子功能与生物过程中显著富集，且MAPK/ERK信号通路呈现显著富集水平。
结论：本研究是首次利用RNA测序技术，结合生物学重复，对BP-3暴露的斑马鱼肠道转录组进行的详细分析。研究结果显示，BP-3暴露组中共存在159个上调基因与73个下调基因；MAPK/ERK信号通路相关条目在分子功能与生物过程中显著富集，基于KEGG分析，MAPK/ERK信号通路呈现显著富集水平。