不同阻滞阶段非梗阻性无精子症的生物信息学分析

doi:10.12280/gjszjk.20220167

摘要/Abstract

摘要：

目的：利用生物信息学筛选不同阻滞阶段非梗阻性无精子症（NOA）相关的调控因子及其调控途径,为后续的功能研究提供依据。方法：从GEO（Gene Expression Omnibus）数据库中获得GSE45885的基因表达谱矩阵,筛选其中的正常样本以及不同阻滞阶段NOA样本,包括4例正常生精样本和20例NOA患者样本,20例NOA患者中有2例患者处于减数分裂前期阻滞阶段（PRE）,7例处于减数分裂期阻滞阶段（MEI）,11例处于减数分裂后期阻滞阶段（POST）。将正常生精样本设置为对照组（CON）,使用GEO2R在线工具鉴定正常生精样本与不同阻滞阶段NOA样本之间的差异基因,对GEO芯片中不同阻滞阶段的NOA数据整合归一化并进行矫正后取交集。对差异表达的基因以及其靶基因进行GO（Gene Ontology）和KEGG（Kyoto Encyclopedia of Genes and Genomes）富集分析,获取关键通路;基于在线工具（STRING）构建蛋白质相互作用（PPI）网络图。然后利用Cytoscape中的CytoHubba插件对枢纽基因进行筛选。结果：在PRE中筛选出463个上调基因,12个下调基因;在MEI和POST中分别发现2个和3个下调基因,无上调基因。PRE中上调基因通过一些重要途径,如精子发生、精子细胞发育、精子的运动、纤毛运动和微管形成等功能来调控生精过程。在上述的3个阻滞阶段NOA中筛选出2个共同差异基因,均为微小RNA（miRNA）,2个miRNA有58个共同靶基因。利用在线生物信息学工具STRING成功构建PRE中上调基因的PPI网络图,并使用Cytoscape筛选出网络中前10个枢纽基因,包括DNAI1、DNAI2、PGK2、ROPN1L、ARMC4、DRC1、DNAAF3、CABYR、ZMYND10和CCDC65。结论：识别枢纽基因和共同差异基因及其通路为后续研究NOA的发生机制提供了有价值的参考,DRC1、ARMC4、MIR15A和MIR509-3可作为后续NOA发病机制研究的潜在靶点。

关键词: 无精子症, 基因表达谱, 非梗阻性无精子症, 靶基因, 功能富集分析

Abstract:

Objective: To screen the regulatory factors and the related pathways of non-obstructive azoospermia (NOA), and to explore the underlying molecular mechanisms by bioinformatics. Methods: The gene expression matrix of GSE45885 was obtained from GEO database. The samples of the NOA at the different arrest stages and the controls were screened, including 20 NOA samples and 4 normal samples. Among the 20 NOA patients, 2 patients were in the premeiotic arrest stage (PRE), 7 patients in the meiotic arrest stage (MEI) and 11 were in postmeiotic arrest stage (POST). The GEO2R online tool was used to identify the differentially expressed genes (DEGs). The data of three arrest stages of NOA in GEO chip were integrated and normalized, and then the intersection was obtained after correction. Gene Ontology（GO）enrichment analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis were performed for the differentially expressed genes and their target genes to obtain key pathways. Protein-protein interaction (PPI) network graph was constructed based on online tool (STRING). Finally, CytoHubba in Cytoscape was used to screen Hub genes. Results: 463 up-regulated genes and 12 down-regulated genes were screened in PRE. Two and three down-regulated genes were found in MEI and POST, respectively. There were no up-regulated genes in MEI and POST. The up-regulated genes in PRE participated in the regulation of spermatogenesis through some important pathways such as spermatogenesis, spermatid development, sperm motility, motile cilium and microtubule formation. Two common differential expression genes were screened out from the above three arrest stages of NOAs, two of them were microRNAs (miRNA), and two miRNAs had 58 common target genes. An online bioinformatics tool STRING was used to successfully construct a PPI network map of up-regulated genes in PRE, and Cytoscape was used to screen out the top 10 Hub genes in the network including DNAI1, DNAI2, PGK2, ROPN1L, ARMC4, DRC1, DNAAF3, CABYR, ZMYND10, CCDC65. Conclusions: The identification of Hub genes, common differential genes and their pathways will provide valuable references for the subsequent study on the mechanism of NOA. DRC1, ARMC4, MIR15A and MIR509-3 may serve as potential targets for subsequent studies on the etiological mechanism of NOA.

Key words: Azoospermia, Gene expression profiling, Non-obstructive azoospermia, Target gene, Functional enrichment analysis

王万伦, 刘桐佳, 张婷, 李硕, 边艳超, 肖瑞. 不同阻滞阶段非梗阻性无精子症的生物信息学分析[J]. 国际生殖健康/计划生育, 2022, 41(4): 270-274.

WANG Wan-lun, LIU Tong-jia, ZHANG Ting, LI Shuo, BIAN Yan-chao, XIAO Rui. Bioanalysis of Non-Obstructive Azoospermia at Different Arrest Stages[J]. Journal of International Reproductive Health/Family Planning, 2022, 41(4): 270-274.

图/表 6

图1 NOA数据归一化矫正后注：CON 正常生精样本,NOA 非梗阻性无精子症患者样本。

图2 NOA数据集可视化处理注：A 3个阻滞阶段NOA差异基因数量,B 3个阻滞阶段NOA下调基因取交集,C 2个下调miRNA靶基因取交集。

图3 MIR15A和MIR509-3的共同靶基因

图4 PRE组上调基因GO富集分析注：BP 生物过程,CC 细胞成分。

表1 共同靶基因的GO及KEGG信号通路富集分析

种类	序列号	功能及通路描述	P
BP	GO:0007156	通过质膜黏附分子黏附同质细胞	5.44×10^-17
BP	GO:0007399	神经系统发育	2.33×10^-13
BP	GO:0007155	细胞黏附	1.83×10^-8
CC	GO:0005887	质膜的组成部分	5.72×10^-5
MF	GO:0005509	钙离子结合	2.15×10^-11
PATHWAY	hsa00562	磷酸肌醇代谢	1.29×10^-2
PATHWAY	hsa04070	磷脂酰肌醇信号系统	2.38×10^-2
PATHWAY	hsa04261	心肌细胞肾上腺素能信号传导	4.46×10^-2

图5 PRE组上调基因的PPI网络

参考文献 21

[1]	Ferlin A, Foresta C. New genetic markers for male infertility[J]. Curr Opin Obstet Gynecol, 2014, 26(3):193-198. doi: 10.1097/GCO.0000000000000061. doi: 10.1097/GCO.0000000000000061 URL
[2]	Khourdaji I, Lee H, Smith RP. Frontiers in hormone therapy for male infertility[J]. Transl Androl Urol, 2018, 7(Suppl 3):S353-S366. doi: 10.21037/tau.2018.04.03. doi: 10.21037/tau.2018.04.03 URL
[3]	Ge P, Zhang J, Zhou L, et al. CircRNA expression profile and functional analysis in testicular tissue of patients with non-obstructive azoospermia[J]. Reprod Biol Endocrinol, 2019, 17(1):100. doi: 10.1186/s12958-019-0541-4. doi: 10.1186/s12958-019-0541-4 URL
[4]	Gu X, Li H, Chen X, et al. PEX10, SIRPA-SIRPG, and SOX5 gene polymorphisms are strongly associated with nonobstructive azoospermia susceptibility[J]. J Assist Reprod Genet, 2019, 36(4):759-768. doi: 10.1007/s10815-019-01417-w. doi: 10.1007/s10815-019-01417-w URL
[5]	Bai G, Zhai X, Liu L, et al. The molecular characteristics in different procedures of spermatogenesis[J]. Gene, 2022, 826:146405. doi: 10.1016/j.gene.2022.146405. doi: 10.1016/j.gene.2022.146405 URL
[6]	Arafat M, Har-Vardi I, Harlev A, et al. Mutation in TDRD9 causes non-obstructive azoospermia in infertile men[J]. J Med Genet, 2017, 54(9):633-639. doi: 10.1136/jmedgenet-2017-104514. doi: 10.1136/jmedgenet-2017-104514 pmid: 28536242
[7]	Liu L, Li F, Wen Z, et al. Preliminary investigation of the function of hsa_circ_0049356 in nonobstructive azoospermia patients[J]. Andrologia, 2020, 52(11):e13814. doi: 10.1111/and.13814. doi: 10.1111/and.13814
[8]	Majzoub A, Arafa M, Khalafalla K, et al. Predictive model to estimate the chances of successful sperm retrieval by testicular sperm aspiration in patients with nonobstructive azoospermia[J]. Fertil Steril, 2021, 115(2):373-381. doi: 10.1016/j.fertnstert.2020.08.1397. doi: 10.1016/j.fertnstert.2020.08.1397 pmid: 33059887
[9]	Wu X, Lin D, Sun F, et al. Male Infertility in Humans: An Update on Non-obstructive Azoospermia (NOA) and Obstructive Azoospermia (OA)[J]. Adv Exp Med Biol, 2021, 1288:161-173. doi: 10.1007/978-3-030-77779-1_8. doi: 10.1007/978-3-030-77779-1_8
[10]	Kasak L, Laan M. Monogenic causes of non-obstructive azoospermia: challenges, established knowledge, limitations and perspectives[J]. Hum Genet, 2021, 140(1):135-154. doi: 10.1007/s00439-020-02112-y. doi: 10.1007/s00439-020-02112-y URL
[11]	Oka S, Shiraishi K, Matsuyama H. Effects of human chorionic gonadotropin on testicular interstitial tissues in men with non-obstructive azoospermia[J]. Andrology, 2017, 5(2):232-239. doi: 10.1111/andr.12292. doi: 10.1111/andr.12292 pmid: 27860441
[12]	Zhang J, He X, Wu H, et al. Loss of DRC1 function leads to multiple morphological abnormalities of the sperm flagella and male infertility in human and mouse[J]. Hum Mol Genet, 2021, 30(21):1996-2011. doi: 10.1093/hmg/ddab171. doi: 10.1093/hmg/ddab171 URL
[13]	Gao Y, Xu C, Tan Q, et al. Case Report: Novel Biallelic Mutations in ARMC4 Cause Primary Ciliary Dyskinesia and Male Infertility in a Chinese Family[J]. Front Genet, 2021, 12:715339. doi: 10.3389/fgene.2021.715339. doi: 10.3389/fgene.2021.715339 URL
[14]	Liu L, Jyu J, Wang X, et al. Progress of miRNA in male infertility: how close are we to noninvasive and accurate diagnostic markers? Systematic review and meta-analysis[J]. Biomark Med, 2021, 15(17):1681-1692. doi: 10.2217/bmm-2020-0434. doi: 10.2217/bmm-2020-0434
[15]	Tektemur A, Etem Önalan E, Kaya Tektemur N, et al. Verapamil-induced ion channel and miRNA expression changes in rat testis and/or spermatozoa may be associated with male infertility[J]. Andrologia, 2020, 52(10):e13778. doi: 10.1111/and.13778. doi: 10.1111/and.13778
[16]	Sirotkin AV, Kisová G, Brenaut P, et al. Involvement of microRNA Mir15a in control of human ovarian granulosa cell proliferation, apoptosis, steroidogenesis, and response to FSH[J]. Microrna, 2014, 3(1):29-36. doi: 10.2174/2211536603666140227232824. doi: 10.2174/2211536603666140227232824 URL
[17]	Xu P, Wang Y, Deng Z, et al. MicroRNA-15a promotes prostate cancer cell ferroptosis by inhibiting GPX4 expression[J]. Oncol Lett, 2022, 23(2):67. doi: 10.3892/ol.2022.13186. doi: 10.3892/ol.2022.13186 URL
[18]	Cannarella R, Barbagallo F, Crafa A, et al. Seminal Plasma Transcriptome and Proteome: Towards a Molecular Approach in the Diagnosis of Idiopathic Male Infertility[J]. Int J Mol Sci, 2020, 21(19):7308. doi: 10.3390/ijms21197308. doi: 10.3390/ijms21197308 URL
[19]	Ozawa H. Kisspeptin neurons as an integration center of reproductive regulation: Observation of reproductive function based on a new concept of reproductive regulatory nervous system[J]. Reprod Med Biol, 2022, 21(1):e12419. doi: 10.1002/rmb2.12419. doi: 10.1002/rmb2.12419
[20]	Maekawa M, Ito C, Toyama Y, et al. Localisation of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse[J]. Andrologia, 2011, 43(3):180-188. doi: 10.1111/j.1439-0272.2010.01049.x. doi: 10.1111/j.1439-0272.2010.01049.x pmid: 21486398
[21]	Yamada D, Yoshida M, Williams YN, et al. Disruption of spermatogenic cell adhesion and male infertility in mice lacking TSLC1/IGSF4, an immunoglobulin superfamily cell adhesion molecule[J]. Mol Cell Biol, 2006, 26(9):3610-3624. doi: 10.1128/MCB.26.9.3610-3624.2006. doi: 10.1128/MCB.26.9.3610-3624.2006 URL