幼羊和成年羊颅骨缝线处硬脑膜组织中差异表达基因的筛选及其生物学功能分析

Screening of differentially expressed genes in dura mater at the skull suture of young and adult sheep and their bioinformatics analysis

目的通过生物信息学方法寻找幼羊和成年羊颅骨缝线处硬脑膜差异表达的基因,分析影响颅骨生长的基因及其对应的信号通路。方法取6只51日龄幼羊和6只12月龄成年羊顶额缝及矢状缝处硬脑膜标本,进行基因测序。以成年羊为对照组,幼羊为实验组,对结果行表达差异性分析,对差异表达的基因行GO富集分析,再对影响颅骨生长的上调及下调基因集分别行KEGG信号通路富集分析。取冻存的幼羊和成年羊硬脑膜组织,采用荧光定量PCR方法对差异表达最高的前10位基因进行验证。结果与成年羊相比,幼羊硬脑膜组织中的差异表达基因共765个,其中上调基因417个,下调基因348个。对幼羊与成年羊硬脑膜组织中差异表达上调基因富集后可见骨的矿化这一富集功能,对差异表达下调基因集富集后可见骨的矿化正向调节这一富集功能。在上调基因中,骨的矿化包含15个基因,分别为ENPP1、HIF1A、IBSP、MMP-13、PTN、OMD、COL1A2、BGLAP、FBN2、ASPN、TMEM119、RFLNB、PHOSPHO1、LOX、ISG15;在下调基因中,骨的矿化正向调节包含5个基因,分别为ISG15、OSR1、BMP-4、BMP-6、BMP-7。KEGG信号通路分析显示,骨的矿化相关基因主要涉及PI3K/Akt信号通路,骨的矿化正向调节相关基因主要涉及TGF-β信号通路。在上调基因中选取上调最多的前6位基因COL1A2、MMP13、PHOSPHO1、IBSP、OMD、PTN和下调基因中下调最多的前2位基因BMP4、BMP6进行荧光定量PCR检测,结果显示同一缝线处幼羊和成年羊上述差异基因表达差异均有统计学意义(P均<0.05)。结论生物信息学分析显示,幼羊和成年羊颅骨缝线处硬脑膜组织中存在大量差异表达的基因,涉及骨的矿化和骨的矿化调节;骨的矿化基因可能通过PI3K/Akt信号通路来促进颅骨的生长,骨形态蛋白基因可能通过TGF-β信号通路来调控缝线的闭合。

Objective Using bioinformatics methods to find genes that are differentially expressed in the dura mater at the skull suture of the young and adult sheep,and to analyze the genes that affect the growth of the skull and their corresponding signal pathways.Methods We took the dura mater of the apical frontal suture and sagittal suture for genetic sequencing from six 51-day-old young sheep and six 12-month-old adult sheep.With adult sheep as the control group and young sheep as the experimental group,the results were analyzed for the differential expression,and the differentially expressed genes were analyzed for GO enrichment.Then,the up-regulated and down-regulated genes that affected the growth of the skull were enriched by the KEGG signaling pathway.Frozen young sheep and adult sheep dura mater tissues were taken,and the top 10 genes with the highest differential expression were verified by fluorescence quantitative PCR.Results Compared with the adult sheep,there were 765 differentially expressed genes in the dural tissues of young sheep,including 417 up-regulated genes and 348 down-regulated genes.Bone mineralization was seen after the enrichment of differentially expressed genes in the dural tissues of young sheep and adult sheep,and the positive regulation of bone mineralization was seen after the enrichment of differentially expressed genes.among the up-regulated genes,bone mineralization included 15 genes,namely ENPP1,HIF1 A,IBSP,MMP13,PTN,OMD,COL1 A2,BGLAP,FBN2,ASPN,TMEM119,RFLNB,PHOSPHO1,LOX,and ISG15;among the down-regulated genes,the positive regulation of bone mineralization included 5 genes,namely ISG15,OSR1,BMP4,BMP6,and BMP7.KEGG signaling pathway analysis showed that bone mineralizationrelated genes mainly involved PI3 K-Akt signaling pathway,and positive regulation ofbone mineralization genes mainly involved TGF-β signaling pathway.Among the up-regulated genes,the first 6 genes with the most up-regulation were COL1 A2,MMP13,PHOSPHO1,IBSP,OMD,and PTN,and the top 2 genes with the most down-regulation were BMP4 and BMP6,which were then selected for fluorescence quantitative PCR detection.The results showed that the difference in the abovementioned differentially expressed genes in the same suture between the young sheep and adult sheep was statistically significant(P < 0.05).Conclusion Bioinformatics analysis showed that there are a large number of differentially expressed genes in the dura mater tissues at the suture of the young and adult sheep,which are involved in the bone mineralization and bone mineralization regulation;bone mineralization genes may promote the growth of the skull through the PI3 K-Akt signaling pathway,and bone morphogenetic protein genes may regulate the closure of sutures through the TGF-β signaling pathway.