基于生物信息学技术的非梗阻性无精子症关键microRNAs和基因的研究

Research on key microRNAs and genes of non-obstructive azoospermia based on bioinformatics technology

目的应用生物信息学方法研究非梗阻性无精子症(NOA)的关键微小RNA(microRNAs,miRNAs)和差异表达基因,为非梗阻性无精子症的病因分析提供新的思路。方法在PubMed、Embase和Web of Science中筛选、提取并整合文献报道的NOA相关的miRNAs,并利用联川生物云平台预测靶基因。检索NCBIGEO数据库中的非梗阻性无精子症基因芯片数据,获得GSE145467和GSE108886数据集并进行GEO2R在线分析,得出NOA相关的差异表达基因,并与预测的靶基因取交集获得最终的差异表达基因。运用DAVID对差异表达基因进行GO富集和KEGG通路分析,应用STRING构建差异表达基因相关的蛋白质互作网络。结果共检索到5条差异表达miRNAs,其中上调表达1条为miR-10b-5p,下调表达4条分别为miR-34b-5p、miR-34b-3p、miR-34c-5p和miR-449a。共得出最终差异表达基因868个,其中上调基因776个,下调基因92个。GO富集结果显示,差异基因参与的生物过程(BP)主要包括纤毛组装、精子轴丝组装、纤毛依赖性细胞运动、顶体组装、精子发生、细胞蛋白质代谢过程等;细胞组成(CC)主要包括活动纤毛、轴丝、精子鞭毛、中心粒等;分子功能(MF)主要包括蛋白质结合、纤毛蛋白轻链的结合和蛋白质稳定化等。KEGG相关通路涉及卵巢类固醇激素生成、多个物种长寿调控途径、扩张型心肌病、内分泌抵抗、孕酮介导的卵母细胞成熟等。通过cytoscape分析前20位的hub基因分别为TEKT3、EFHC1、DYNLL2、DNAH2、CETN1、SPATA7、ASRGL1、CCDC146、PLCZ1、SPAG16、DNAL1、EFCAB11、SPA4L、LIN7A、TEKT1、FXR1、RPGRIP1、DPY19L2、DDX25、ZC3H14。结论本研究鉴定的miRNAs、hub基因和相关通路,在精子发生过程中发挥着重要的作用,可为后续拓展研究NOA的病因机制提供参考靶点。

Objective:To study the key miRNAs and differentially expressed genes in non-obstructive azoospermia(NOA)by applying bioinformatics methods,and provide new ideas for the diagnosis and treatment of NOA.Methods:The literatures related NOA associated miRNAs were screened from PubMed,Embase and Web of Science,as well as extracted and integrated.The target genes were predicted by using Lianchuan Omicstudio.The gene microarray data of non-obstructive azoospermia in NCBIGEO database were retrieved,and the datasets of GSE145467 and GSE108886 were obtained and conducted analysis online by GEO2R to derive NOA-related differentially expressed genes.The final differentially expressed genes were obtained by intersection with the predicted target genes.GO enrichment and KEGG pathway analysis of the differentially expressed genes were performed by using DAVID.STRING was applied to construct the interaction network of the differentially expressed gene-related protein.Results:Five differentially expressed miRNAs were identified,of which one with up-regulated expression was miR-10b-5p,and four with down-regulated expression were miR-34b-5p,miR-34b-3p,miR-34c-5p,and miR-449a.A total of 868 differentially expressed genes were identified through analysis of GEO database data,including 776 up-regulated genes and 92 down-regulated genes.GO enrichment analysis showed that the biological processes(BP)in which differential genes were involved mainly included cilia assembly,sperm axoneme assembly,cilia-dependent cell motility,acrosome assembly,spermatogenesis,and cellular protein metabolic processes.Cell composition(CC)mainly included active cilia,axonemes,sperm flagella,centrioles,etc.Molecular functions(MF)mainly included protein binding,binding of ciliary protein light chains,etc.KEGG enrichment analysis pathways involved in ovarian steroid hormone production,multiple species longevity regulatory pathways,dilated cardiomyopathy,endocrine resistance,progesterone-mediated oocyte maturation,etc.The top 20 hub genes analyzed by cytoscape were TEKT3,EFHC1,DYNLL2,DNAH2,CETN1,SPATA7,ASRGL1,CCDC146,PLCZ1,SPAG16,DNAL1,EFCAB11,SPA4L,LIN7A,TEKT1,FXR1 RPGRIP1,DPY19L2,DDX25,ZC3H14.Conclusions:The miRNAs,hub genes and related pathways identified in this study may play an important role in spermatogenesis,which can provide reference targets for further studies on the etiological mechanisms of NOA.