视黄酸诱导的小鼠神经管畸形基因表达趋势分析

Trend analysis of differentially expressed genes in retinoic acid-induced neural tube defects in mouse model

目的·探究小鼠胚胎在视黄酸(retinoic acid,RA)诱导下产生神经管畸形的分子调控机制,揭示小鼠神经管闭合阶段基因表达规律。方法·基于已获得的小鼠胚胎神经管闭合关键期[胚胎发育第8.5日(embryonic day 8.5,E8.5)、E9.5、E10.5]高质量脑泡转录组数据,利用短时间序列表达挖掘器(Short Time-series Expression Miner,STEM)软件分别得到RA处理组和正常组在3个时间点的基因表达趋势数据。对处理组与正常组基因表达趋势不一致的基因进行基因本体(Gene Ontology,GO)富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,并随机筛选候选基因以验证测序数据可靠性。利用RA诱导构建神经管畸形小鼠模型,分为处理组和正常组,每组各9只。处理组和正常组孕鼠在E7.5分别接受28 mg/kg RA和香油灌胃处理,在E8.5、E9.5、E10.5收集胎鼠脑泡组织,对筛选的候选基因进行实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)验证。结果·正常组共检测出18255个基因的表达量数据,处理组共检测出19037个基因的表达量数据;正常组基因可归纳至7个具有显著意义的表达模式中,处理组基因可归纳至6个具有显著意义的表达模式中;正常组和处理组检测到表达的基因数目足够、表达的模式相似,具有可比性。进一步分析发现正常组中呈现上升表达趋势但在处理组中呈现下降表达趋势的基因共有46个,在生物学过程层面富集在器官发育、神经元凋亡的正负调控、少突胶质细胞增殖、成纤维生长因子信号通路等;在细胞组分层面,主要参与组成细胞、神经元的基本结构;在分子功能层面,主要与成纤维细胞生长因子受体结合有关。正常组中呈现下降表达趋势而在处理组中呈现上升表达趋势的61个基因,在生物学过程层面富集在细胞溶解、氨基酸/离子转运等功能上;在细胞组分层面,富集在胞内分子、皮质颗粒、胞外区域、细胞间隙等;在分子功能层面,与一系列酶及转运蛋白的活性有关。RT-qPCR验证结果显示转录组测序数据真实可靠。结论·RA干预使小鼠胚胎发育过程中发生基因表达失调和应激反应,导致胚胎发育异常,机体自我保护相关信号通路激活,维持胚胎正常发育的基因受到抑制。

Objective·To explore the molecular regulatory mechanism of neural tube defect(NTD)induced by retinoic acid(RA)in mouse embryos,and reveal the gene expression regularity of neural tube closure in mice.Methods·Based on the high-quality brain vesicle transcriptome data of mouse embryo during the critical period of neural tube closure[embryonic day 8.5(E8.5),E9.5 and E10.5],the gene expression trend data of the NTD group and the control group were obtained by using Short Time-series Expression Miner(STEM)software.Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed for genes with different expression trends between the NTD group and the control group.Some candidate genes were screened for validation.Pregnant mice were divided into the NTD group and control group,with 9 mice in each group.Pregnant mice in the NTD group were treated with RA and those in the control group were treated with sesame oil by gavage at E7.5.Foetal rat brain vesicle tissues were collected at E8.5,E9.5 and E10.5 for experiments.Based on the above animal tissues,the screened candidate genes were validated by quantitative real-time PCR(RT-PCR).Results·A total of 18255 genes were detected in the transcriptome data of the control group,and the expression patterns of these genes could be summarized into 7 significant profiles.A total of 19037 gene expression data were detected in the transcriptome data of the NTD group,and gene expression patterns could be summarized into 6 profiles with significant significance.A total of 46 genes in the control group showed an upward trend but a downward trend in the NTD group.They were enriched in the positive and negative regulation of organ development,neuronal apoptosis,oligodendrocyte proliferation,and fibroblast growth factor signaling pathway at the biological process level.At the cellular component level,they were mainly involved in the basic structure of cells and neurons;At the molecular functional level,they were mainly related to the binding of fibroblast growth factor receptor.A total of 61 genes showed a downward trend in the control group but an upward trend in the NTD group.These genes were enriched in functions such as cell lysis and amino acid/ion transport at the biological process level.At the cellular component level,they were enriched in intracellular molecules,particles,extracellular region,intercellular space,etc.At the molecular function level,they were related to the activity of a series of enzymes and transporters.The results of RT-qPCR showed that the transcriptome sequencing data were authentic and reliable.Conclusion·RA intervention causes abnormal cellular activities and stress responses during mouse embryo development,leading to abnormal embryo development,activation of signalling pathways related to organismal self-protection,and suppression of genes that maintain normal embryo development.