先天性巨结肠竞争内源性RNA网络构建和分析

Expression Profile Screening of ceRNA Network Construction for Hirschsprung disease

目的:基于在线数据库,利用生物信息学的方法构建先天性巨结肠(Hirschsprung disease,HSCR)竞争性内源RNA(competitive endogenous RNA,ceRNA)调控网络。方法:从GEO数据库中下载HSCR长链非编码RNA(long non-coding RNA,lncRNA)-seq、微小RNA(microRNA,miRNA)-seq和mRNA-seq,采用limma算法筛选出差异表达lncRNAs、miRNAs和mRNAs。利用R包cluster Profile对差异基因进行GO分析和KEGG分析。利用生物信息学预测网站预测靶点关系,采用Cytoscape v3.5.1软件构建可视化ceRNA网络。利用实时荧光定量聚合酶链反应,免疫组织化学及免疫荧光进行临床样本验证。采用t检验进行统计学分析。结果:从HSCR样本中筛选出219个上调和116个下调的lncRNAs;61个上调和21个下调的miRNAs;1216个上调的和1376个下调的mRNAs;差异基因主要富集在神经系统形成、免疫反应、凋亡调控等GO功能;涉及PPAR信号通路、Hippo信号通路、cAMP信号通路、Th1和Th2细胞分化、MAPK信号通路、Wnt信号通路等信号通路。在构建的ceRNA网络中,共筛选出37个核心lncRNAs、13个靶向miRNAs及22个具有靶向关系的mRNAs。miR-149-3p呈现出与ENST00000568239.1和X连锁凋亡的抑制剂(X-linked inhibitor of apoptosis,XIAP)相反的表达趋势。结论:构建了参与HSCR发生发展的ceRNA调控网络,为HSCR分子机制研究提供新的靶标和依据。

Objective:To construct the competitive endogenous RNA (ceRNA) regulatory network of Hirschsprung's disease (HSCR) through bioinformatics based upon online databases.Methods:From the GEO database, long non-coding RNA (lncRNA)-seq, microRNA (miRNA)-seq and mRNA-seq data related to HSCR were downloaded. Limma algorithm was employed for screening for differentially expressed lncRNAs, miRNAs and mRNAs. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes & Genomes) analyses of differentially expressed genes were performed with R package clusterProfiler. Bioinformatic prediction websites were utilized for predicting target relationships. And Cytoscape v3.5.1 software was employed for constructing a visualized ceRNA network. Clinical samples were validated with real-time quantitative fluorescent polymerase chain reaction (qPCR), immunohistochemistry and immunofluorescence. Statistical analysis was conducted by t-test.Results:Within HSCR samples, 219 up-regulated and 116 down-regulated lncRNAs, 61 up-regulated and 21 down-regulated miRNAs and 1,216 up-regulated and 1,376 down-regulated mRNAs were identified. The differentially expressed genes were largely enriched in GO functions such as nervous system development, immune response and apoptotic regulation. These genes also participated in signaling pathways of PPAR, Hippo, cAMP, Th1/Th2 cell differentiation, MAPK and Wnt. In constructed ceRNA network, a total of 37 core lncRNAs, 13 target miRNAs and 22 mRNAs with targeting relationships were selected. And miR-149-3p exhibited an opposite expression trend as compared to ENST00000568239.1 and X-linked inhibitor of apoptosis (XIAP).Conclusions:A ceRNA regulatory network involved in the development and progression of HSCR has been constructed for providing novel targets and rationales for elucidating molecular mechanisms of HSCR.