先天性巨结肠lncRNA-mRNA共表达网络构建和分析

Construction and analysis of lncRNA-mRNA co-expression network for Hirschsprung disease

目的通过生物信息学的方法构建先天性巨结肠(Hirschsprung disease,HSCR)调控网络。方法RNA测序检测HSCR组织和正常结肠组织中的长链非编码RNA(long non-coding RNA,lncRNA)和mRNA表达谱。结合GSE96854数据库的mRNA表达谱数据,构建lncRNA-mRNA共表达网络。各选择3个lncRNA和mRNA,采用QPCR验证RNA测序结果。结果RNA测序共筛选出224个lncRNA,其中上调和下调的lncRNA分别为108和116个;785个差异表达的基因(differentially expressed genes,DEGs),其中上调和下调的DEGs分别为82个和703个。DEGs主要富集在cGMP-PKG、NF-κB信号通路等KEGG通路上。在GSE96854数据中,显著上调和显著下调的DEGs分别为1216个和1376个。两个数据集中后共有的DEGs有178个,其中显著下调的DEGs有175个,而显著上调的DEGs仅有3个。蛋白质-蛋白质相互作用(protein-protein interaction,PPI)结果表明,SNAP25、SYP、ATP2B3和ZAP70位于网络中心的基因,与诸多基因尤其是SNAP25有互作关系。共筛选出146对lncRNA-mRNA调控网络。筛选出AC074286.1-ADRA2A-cGMP-PKG和DUX4L9-ATP2B3-NF-κB信号通路的lncRNA-mRNA通路调控网络。实时定量基因扩增荧光检测结果表明ADRA2A、ATP2B3、TNFSF14、AC074286.1、DUX4L9和REXO1L1P在HSCR中显著下调,且与RNA测序结果一致。结论参与炎症反应、细胞凋亡和神经相关的lncRNA和mRNA分子标志物在HSCR中发挥着重要的作用。

Objective To construct a regulatory network of Hirschsprung's disease(HSCR)through bioinformatics.Methods RNA sequencing was utilized for detecting the expression profiles of long non-coding RNA(lncRNA)and mRNA in HSCR and normal colon tissues.A co-expression network of lncRNA-mRNA was constructed with the mRNA expression data from the database of GSE96854.Three lncRNAs and mRNAs were selected for validation using quantitative polymerase chain reaction(QPCR).Results A total of 224 lncRNAs were identified by RNA sequencing,including 108 up-regulated and 116 down-regulated lncRNAs;785 differentially expressed genes(DEGs)were identified with 82 up-regulated and 703 down-regulated.DEGs were mainly enriched in KEGG pathways such as cGMP-PKG and NF-κB signaling pathways.In the GSE96854 dataset,1216 significantly up-regulated and 1376 significantly down-regulated DEGs were identified.There were 178 common DEGs between two datasets with 175 significantly down-regulated and only 3 significantly up-regulated DEGs.Protein-protein interaction(PPI)analysis revealed that SNAP25,SYP,ATP2B3 and ZAP70 were core interactive genes in the network,especially SNAP25.A total of 146 lncRNA-mRNA regulatory networks were identified.AC074286.1-ADRA2A-cGMP-PKG and DUX4L9-ATP2B3-NF-κB signaling pathways were identified as lncRNA-mRNA pathway regulatory networks.QPCR results revealed a significant down-regulation of ADRA2A,ATP2B3,TNFSF14,AC074286.1,DUX4L9 and REXO1L1P,consistent with the results of RNA sequencing.Conclusion LncRNA and mRNA molecular markers are closely involved in inflammation,apoptosis and neural-related processes of HSCR.