基于RNA-Seq技术的先天性巨结肠转录组学

Screening of differentially expressed genes in Hirschsprung disease based on RNA-Seq technology

目的筛选先天性巨结肠(hirschsprung disease,HSCR)患者病变肠组织和正常肠组织的差异表达基因.方法选取2例全结肠型HSCR、1例长段型HSCR患者,从福尔马林固定后石蜡包埋样本中提取RNA,采用去除核糖体的方法构建cDNA文库后,应用RNA-Seq全面分析3对肠组织样本的转录组,筛选出病变组织和正常组织的差异表达基因.应用GO(gene ontology)方法分析差异表达基因的功能聚类,应用KEGG(kyoto encyclopedia of genes and genomes)信号通路富集性方法分析差异表达基因相关的信号通路.结果 3对标本的总RNA提取质量合格,RNA-Seq测序数据质量合格,测序数据与人类基因组参考基因对比匹配率合格,样品间基因表达相关性合格.3对病变组织和正常组织共有1382个差异表达基因,包括899个表达一致上调的基因、483个表达一致下调的基因.1382个差异表达基因被分为383个功能聚类.KEGG分析表明这些差异表达基因参与胆汁分泌、蛋白质消化与吸收、脂肪消化与吸收、维生素A代谢、类固醇激素生物合成、淀粉和蔗糖代谢等信号通路.结论本研究首次利用RNA-Seq技术对先天性巨结肠病变组织和正常组织中的差异表达基因进行了全面分析,可为推动疾病发病机制的研究提供新思路.

AIM To screen the differentially expressed genes（DEGs） in aganglionic and ganglionated colon tissues of hirschsprung disease（HSCR） patients.METHODS RNA was extracted from paired-colon samples（formalin-fixed, paraffin-embedded sections） of three HSCR patients（two affected by total colonic aganglionosis and one by long-segment HSCR） and purified. Ribosomal RNA was eliminated before generating the cDNA library. After RNA-sequencing, the gene ontology（GO） analysis was performed to uncover the biological functions of DEGs. The kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analysis was applied to find out the relatedsignal pathways.RESULTS There were a total of 1382 significantly commom DEGs in the three paired samples, including 899 consistently up-regulated DEGs and 483 consistently down-regulated DEGs. The 1382 DEGs were categorized into 383 functional categories by GO analysis. The KEGG pathway analysis suggested 25 significant signal pathways, including bile secretion, protein digestion and absorption, fat digestion and absorption, retinol metabolism, steroid hormone biosynthesis, and starch and sucrose metabolism.CONCLUSION In the current study, the DEGs of aganglionic and ganglionated colon tissues were thoroughly analyzed for the first time, which will shed new light on the pathogenesis of HSCR.