宫颈癌阴道灌洗液源外泌体差异表达miRNA筛选及其关键靶基因的生物信息学分析  

Screening of differentially expressed miRNAs in exosomes from vaginal lavage fluid in patients with cervical cancer and bioinformatics analysis of target genes

作　　者：吕单单 徐梦伟 王磊[2] 木叶斯尔·买买提 林晨[1] LYU Dandan;XU Mengwei;WANG Lei;Muyesier Maimaiti;LIN Chen(不详;School of Basic Medicine,Xinjiang Medical University,Urumqi 8300172,China)

机构地区：[1]新疆医科大学基础医学院,乌鲁木齐8300172 [2]新疆医科大学公共卫生学院

出　　处：《山东医药》2022年第29期15-19,共5页Shandong Medical Journal

基　　金：新疆维吾尔自治区自然科学基金青年基金项目(2021D01C292)。

摘　　要：目的筛选宫颈癌阴道灌洗液源外泌体差异表达miRNA并通过生物信息学方法分析其关键靶基因。方法收集宫颈癌及宫颈炎患者阴道灌洗液标本,超速离心法提取外泌体,透射电镜(TEM)观察外泌体形态、纳米颗粒追踪分析(NTA)鉴定外泌体粒径大小。将提取的外泌体样本制作基因芯片,采用R语言分析宫颈癌外泌体差异表达miRNA,通过mirtarBase数据库对宫颈癌外泌体差异表达miRNA进行靶基因预测,使用R语言对靶基因进行GO生物功能、KEGG通路富集分析。运用STRING在线工具对差异表达miRNA靶基因进行蛋白质-蛋白质互相作用(PPI)网络分析,Cytoscap对PPI网络进行拓扑,筛选出具有高度连通性的枢纽靶基因。采用R语言对枢纽靶基因进行KEGG信号通路富集分析,与差异表达miRNA靶基因富集的KEGG信号通路取交集,筛选出共有的关键信号通路及与之相关的关键靶基因。结果TEM可观察到典型的外泌体形态,NTA显示样本粒径大小均符合外泌体的粒径标准。宫颈癌患者阴道灌洗液源外泌体中显著差异表达的miRNA共有84个,在差异倍数前20的miRNA中共有miR-320d、miR-320c、miR-24-3p等18个上调miRNA及miR-3613-3p、miR-4668-5p 2个下调miRNA。通过mirtarBase数据库获得与差异表达miRNA相关的273个靶基因,GO生物功能分析显示,差异表达miRNA靶基因主要分布于细胞黏附斑、细胞-基质黏附结点等,主要富集的生物过程为角蛋白结合、泛素样蛋白连接酶结合等,主要富集的分子功能为核糖核蛋白复合物的生物生成、病毒基因表达等;KEGG信号通路分析显示,差异表达miRNA靶基因主要富集的信号通路为癌症中的蛋白聚糖、细胞凋亡等。PPI网络及Cytoscap分析显示,宫颈癌关键信号通路为AMPK信号通路、mTOR信号通路、细胞凋亡、癌症中的蛋白聚糖、TNF信号通路、FoxO信号通路、PD-L1在肿瘤中的表达及PD-1检测点通路,关键靶基因为PIK3R1、EDObjective To screen the differentially expressed miRNAs in exosomes from vaginal lavage fluid in pa-tients with cervical cancer and to analyze their key target genes by bioinformatic method.Methods Specimens of vaginal lavage fluid from patients with cervical cancer and from patients with cervicitis were collected,exosomes were extracted by ultracentrifugation,and exosome morphology was observed by Transmission Electron Microscopy(TEM),and exosome particle size was identified by Nanoparticle Tracking Analysis(NTA).We extracted exosomal samples to make gene chips,and the differentially expressed miRNAs of cervical cancer exosomes were analyzed by R language,and the target genes were predicted by mirtarBase database,and the GO biofunction and KEGG pathway enrichment analysis of the target genes were performed by R language.Protein-protein interaction(PPI)network analysis of differentially expressed miRNA target genes was performed using STRING online tool,and topology of PPI network was performed by Cytoscap to screen out the pivotal target genes with high connectivity.The KEGG signaling pathway enrichment analysis of the hub target genes was performed using R language,and the KEGG signaling pathway enriched with differentially expressed miRNA tar-get genes was intersected to screen out the shared key signaling pathways and the key target genes associated with them.Results Typical exosome morphology could be observed by TEM,and NTA showed that the particle size of the samples all met the particle size criteria for exosomes.A total of 84 miRNAs were significantly differentially expressed in vaginal la-vage-derived exosomes from cervical cancer patients.Eighteen up-regulated miRNAs,including miR-320d,miR-320c and miR-24-3p,and 2 down-regulated miRNAs,including miR-3613-3p and miR-4668-5p,were found among the top 20 differential ploidy miRNAs.GO biofunctional analysis showed that the differentially expressed miRNA target genes were mainly distributed in cell adhesion spots,cell-matrix adhesion junctions,etc.The main e

关 键 词：宫颈癌 外泌体 阴道灌洗液 MIRNA 靶基因 

分 类 号：R737.33[医药卫生—肿瘤]