弓形虫毒力相关效应分子ROP16Ⅲ诱导人肺腺癌A549细胞的基因表达谱  

Gene expression profiling of human lung adenocarcinoma of A549 cells induced by Toxoplasma virulence-related effector ROP16Ⅲ

作　　者：苏雅静 乔霞 汪澎涛 康宇婷 杨宁爱 贾伟 赵志军 SU Ya-jing;QIAO Xia;WANG Peng-tao;KANG Yu-ting;YANG Ning-ai;JIA Wei;ZHAO Zhi-jun(Ningxia Key Laboratory of Clinical and Pathogenic Microbiology,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Laboratory Center of Medicine,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院宁夏临床病原微生物重点实验室,银川750004 [2]宁夏医科大学总医院医学实验中心,银川750004

出　　处：《中国寄生虫学与寄生虫病杂志》2021年第4期473-479,486,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：宁夏自然科学基金(2019AAC03227);第四批宁夏青年科技人才托举工程项目(TJGC2019089)。

摘　　要：目的利用生物信息学方法分析刚地弓形虫(Toxoplasma gondii)棒状体蛋白16Ⅲ(ROP16Ⅲ)重组表达载体转染人肺腺癌A549细胞后的基因表达谱,筛选致病候选基因。方法构建真核表达载体pEGFP-N1-ROP16Ⅲ。将A549细胞分为未转染组、pEGFP-N1组、pEGFP-N1-ROP16Ⅲ组,后两组分别用pEGFP-N1、pEGFP-N1-ROP16Ⅲ质粒进行瞬时转染,48 h后收集各组细胞,提取总RNA,纯化后进行芯片杂交分析,筛选差异表达基因(DEG)。将DEG进行基因本体(GO)功能富集分类和京都基因与基因组百科全书(KEGG)通路分析。利用ToppGene从DEG中筛选弓形虫ROP16Ⅲ致病候选基因。利用STRING在线分析软件对候选基因所编码的蛋白质进行蛋白间相互作用(PPI)网络分析。结果pEGFP-N1-ROP16Ⅲ转染A549细胞后共有566个基因表达差异显著,其中382个基因上调,184个基因下调。对DEG进行生物信息学分析,共获得997条GO注释和50条KEGG信号通路。这些DEG参与了多种生物学过程,包括防御反应、I型干扰素信号通路、先天免疫应答、对外界生物刺激的反应等;参与的信号通路主要集中在抗原处理及呈递,细胞质DNA传感通路,Toll样受体信号通路,NOD样受体信号通路等。ToppGene筛选共获得14个弓形虫ROP16Ⅲ致病候选基因,其中C-X-C基序趋化因子配体11(CXCL11)、Toll样受体3(TLR3)、C-C基序趋化因子26(CCL26)、人类白细胞抗原-E(HLA-E)、早幼粒白血病基因(PML)及信号转导子与转录激活因子2(STAT2)等6个基因在弓形虫方面的研究尚未见报道。PPI网络构建分析发现,DEG编码的蛋白质PPI网络由376个节点(蛋白质)和1152条边(相互作用关系)组成,STAT2、HLA-E和PML位于PPI网络的中心节点处,删除这些关键节点后PPI网络结构涣散。通路富集分析结果显示,CXCL11等6个基因共涉及12条信号通路,主要与趋化因子信号通路、Toll样受体信号通路、程序性坏死、内吞作用及细胞因子-细胞因子受体相互作Objective To analyze the gene expression profile in the A549 human lung adenocarcinoma cell line transfected with the expression vector of Toxoplasma gondii rhoptry protein 16Ⅲ(ROP16Ⅲ),using bioinformatics methods,in order to identify candidate pathogenic genes.Methods The eukaryotic expression vector pEGFP-N1-ROP16Ⅲwas constructed.The A549 cells were assigned to three groups:untransfected,pEGFP-N1 and pEGFP-N1-ROP16Ⅲgroups.Cells in the latter two groups were instantantaneously transfected with pEGFP-N1 and pEGFP-N1-ROP16 plasmid,respectively.After 48 h,the cells were harvested and RNA was extracted.After RNA purification,microarray hybridization analysis was performed to identified differentially expressed genes(DEGs)which were further analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)for functional enrichment classification and pathway analysis.ToppGene was used to screen for Toxoplasma ROP16Ⅲpathogenic candidate genes from DEGs.The STRING online analysis software was used to analyze the protein-protein interaction(PPI)network for protein products of the candidate genes.Results A total of 566 genes were differentially expressed in A549 transfected with Toxoplasma ROP16Ⅲ,of which 382 genes were up-regulated and 184 genes down-regulated.Bioinformatics analysis of the DEGs revealed 997 GO annotations and 50 KEGG signaling pathways.The DEGs were significantly enriched in defense response,type I interferon signaling pathway,innate immune response,and response to external biotic stimulus.KEGG pathway enrichment analysis suggested the DEGs were most significantly enriched in antigen processing and presentation,cytosolic DNA-sensing pathway,Toll-like receptor signaling pathway,and NOD-like receptor signaling pathway.In addition,14 candidate genes were screened out from the DEGs by the ToppGene Suite.Among them,C-X-C motif chemokine 11(CXCL11),toll-like receptor 3(TLR3),C-C motif chemokine ligand 26(CCL26),human leukocyte antigen E(HLA-E),promyelocytic leukemia protein(PML)and sig

关 键 词：弓形虫ROP16Ⅲ 表达谱 生物信息学 蛋白互作网络 

分 类 号：R382.5[医药卫生—医学寄生虫学]