Objective: To investigate the mechanisms of lncRNA on the occurrence and development of NOA by constructing ceRNA regulation network of lncRNA, miRNA and mRNA. Methods: Samples of adult human testis were obtained from NOA patients and OA patients with normal spermatogenesis (controls), recruited from the Reproductive Medicine Center of Nanfang Hospital from June 2017 to June 2018. Differentially expressed lncRNAs and mRNAs in testicular tissues from patients with NOA were identified by microarray analysis in previous association study. In this study, differentially expressed lncRNAs and mRNA were used to construct the ceRNA regulatory network in NOA and clarify the interaction relationship among lncRNA, miRNA and mRNA. GeneMANIA database was used to construct Protein-Protein Interaction (PPI) of the mRNAs in ceRNA regulatory network. WebGestalt toolkit was employed to perform gene function and pathway enrichment analyses of those coding genes. Finally, qRT-PCR and dual luciferase reporter system were employed for further experimental validation. Results: The ceRNA regulatory network of lncRNA, miRNA and mRNA consists of 21 nodes and 26 edges, of which 4 lncRNAs, 13 miRNAs and 4 mRNAs. 19 proteins were found to interact with the mRNA coding proteins in ceRNA regulatory network by PPI analysis. Gene oncology and KEGG pathway enrichment analyses indicate these coding genes were significantly enriched in pentose metabolic process and pentose phosphate pathway. Furthermore, lncRNA ANXA2P3 was found binding with miR-613 and miR-206 to inhibit mRNA TKT expression. Conclusion: lncRNAs exert an important role in the occurrence and development of NOA via ceRNA regulatory network, which could be used as new biomarkers for NOA treatment.
目的： 通过构建长链非编码RNA（lncRNA）、microRNA（miRNA）、mRNA参与的竞争性内源RNA（ceRNA）网络，进一步探究lncRNA在非梗阻性无精子症（NOA）发生发展过程中的调控机制。 方法： 收集2017年6月至2018年6月南方医科大学南方医院生殖中心收治的无精子症患者的睾丸组织。本课题组前期使用NOA患者（试验组）和生精正常的梗阻性无精子症（OA）患者（对照组）的睾丸组织进行基因芯片检测，筛选出差异表达的lncRNA和mRNA。本研究采用在NOA患者中差异表达的lncRNA和mRNA构建NOA ceRNA调控网络，并通过GeneMANIA数据库对ceRNA中编码蛋白的mRNA进行蛋白质-蛋白质互作（PPI）网络构建。然后，采用WebGestalt对PPI中编码蛋白的基因进行功能富集分析。最后，通过实时荧光定量聚合酶链式反应（qRT-PCR）和双荧光素酶报告基因试验进行验证。 结果： NOA的ceRNA调控网络共包括21个节点和26条边，其中4个lncRNA节点，13个miRNA节点以及4个mRNA节点。PPI网络分析发现有19个蛋白与ceRNA网络中mRNA编码蛋白有相互作用。功能富集分析结果显示这些编码蛋白显著性富集于戊糖代谢相关的生物学功能和信号通路。进一步的试验验证，证实lncRNA ANXA2P3通过与miR-613和miR-206竞争性结合，进而抑制mRNA TKT的表达水平。 结论： lncRNA通过ceRNA调控网络影响NOA的发生发展，为NOA的诊治提供了新靶点和治疗方案。.
Keywords: LncRNA; Non-obstructive azoospermia; Regulatory mechanism; ceRNA.