RNA-Seq分析小鼠腭发育关键时期基因的时空表达

RNA-Seq Analysis of Temporal and Spatial Expression of Genes in the Key Period of Palatogenesis in Mice

目的:构建小鼠腭发育时期差异表达基因库(differentially expressed genes bank,DEGB),探究腭发育关键调控因子和调控机制。方法:分别收集C57BL/6孕鼠妊娠13.5、14.5、15.5、16.5d(embryonic day,Ed)胚胎腭板,RNA测序技术筛查差异表达基因(differentially expressed genes,DEGs),选择部分基因进行功能注释,定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)验证和免疫组织化学染色。结果:腭发育第一阶段(Ed13.5～Ed14.5)筛出243个DEGs(27个下调,216个上调);第二阶段(Ed14.5～Ed15.5)筛出208个DEGs(30个下调,178个上调);第三阶段(Ed15.5～Ed16.5)筛出262个DEGs(31个下调,231个上调)。其中微小RNA(micro RNA,miRNA)和晶体蛋白家族多个成员表达变化显著。免疫组织化学结果显示Ed13.5～Ed16.5腭间充质中分泌型磷酸蛋白1(secreted phosphoprotein,Spp1)表达量逐渐升高。结论:miRNA家族、晶体蛋白家族成员以及Spp1基因在腭形成时期可能发挥关键作用。

Objective:To construct DEGB during palatogenesis in mice,and to explore the key regulatory factors and regulation mechanism of palate development.Methods:The embryonic palatal shelves of pregnant C57BL/6at Ed13.5,Ed14.5,Ed15.5,and Ed16.5were collected respectively for RNA-seq to screen DEGs.Selected DEGs were validated and analyzed by qPCR and immunohistochemistry.Results:RNA-seq identified243DEGs(27DEGs were down-regulated and216DEGs were up-regulated)at the first stage of palatal development(Ed13.5-Ed14.5),208DEGs(30DEGs down-regulated and178DEGs up-regulated)at the second stage(Ed14.5-Ed15.5),and262DEGs between E15.5and Ed16.5(31DEGs down-regulated and231DEGs up-regulated)at the third stage(Ed15.5-Ed16.5).The expression of DEGs related with micro RNA and crystallin family changed significantly.Immunohistochemistry showed that Spp1was gradually increased in palatal mesenchyme from Ed13.5to Ed16.5.Conclusion:Members in micro RNA family and crystallin family and Spp1gene play a role in the palatogenesis.