弱精子症精子中miRNAs特异表达及潜在致病靶点的分析

Differentially Expressed Key miRNAs and Potential Targets Analysis in Asthenzoospermia

目的:弱精子症可见于40%的不育男性,其特征是精子活力低下。微小RNA(MicroRNAs,miRNAs)在精子发生中发挥重要作用,但关于精子中miRNAs在弱精子症中的作用知之甚少。本研究试图初探miRNAs在弱精子症的分子机制。方法:收集了重度弱精子症患者和健康男性的精子样本,采用高通量序列技术来识别差异表达的miRNAs,并对差异显著的miRNAs进行生物信息学分析。通过qRT-PCR证实了2个特异性改变的miRNA及其靶基因表达情况。结果:重度弱精子症患者与正常男性相比,共有146个miRNAs(P<0.05;|log2 Fold Change|>1)发生改变,其中表达上调的52个,下调的94个;预测上下调幅度最显著的前10个miRNAs的靶基因,同时在miRDB和TargetScan数据库存在的靶基因共有1407个。富集分析结果显示,miRNAs的靶基因富集于精子细胞的生物过程,还参与精子细胞的氧化代谢、刺激反应、增殖和分化以及凋亡等生物过程。通路分析显示,靶基因可能参与细胞自噬、细胞衰老、PI3K-Akt信号通路、MAPK信号通路、HIF-1信号通路、m TOR信号通路等。其中,在弱精子症精子中特异性上调的hsa-miR-371a-5p和hsa-miR-2355-5p,预测靶基因分别为自噬效应蛋白Beclin1和线粒体内膜蛋白抑素2(prohibitin2,PHB2),二者直接参与线粒体自噬过程。qRT-PCR结果显示随着精子活力的降低,精子中hsa-miR-371a-5p和hsa-miR-2355-5p的表达量升高。结论:本研究发现弱精子症患者精子中特异性失调的miRNAs及其靶基因,为后续深入研究低活力精子中miRNAs参与调控线粒体自噬功能的机制提供新思路和理论依据。

Objective:Asthenozoospermia,a condition observed in 40%of infertile males,is characterized by reduced sperm motility.MicroRNAs(miRNAs)play a pivotal role in spermatogenesis,yet their involvement in asthenozoospermia remains poorly understood.This study aims to elucidate the molecular mechanisms of miRNAs in asthenozoospermia.Methods:Sperm samples were collected from individuals with severe asthenozoospermia and healthy males.High-throughput sequencing was employed to identify differentially expressed miRNAs,followed by bioinformatics analysis of these significant miRNAs.The altered expression of two specific miRNAs and their target genes was confirmed using qRT-PCR.Results:When comparing severe asthenozoospermia patients to healthy males,146 miRNAs exhibited significant alterations(P<0.05;|log2 Fold Change|>1),with 52 upregulated and 94 downregulated miRNAs.The ten most significantly upregulated and downregulated miRNAs were subjected to target gene prediction through the miRDB and TargetScan databases,a total of 1407 target genes were found to be supported by both databases.Enrichment analysis revealed that miRNA target genes were enriched in sperm cell processes,and they were involved in oxidative metabolism,stimulus response,proliferation,differentiation,and apoptosis in sperm cells.Pathway analysis indicated that target genes might participate in processes such as autophagy,cellular senescence,the PI3K-Akt signaling pathway,the MAPK signaling pathway,the HIF-1 signaling pathway,and the mTOR signaling pathway.Notably,among the specifically upregulated miRNAs in asthenozoospermia,hsa-miR-371a-5p and hsa-miR-2355-5p were identified,with their respective target genes being the autophagy effector protein Beclin1 and mitochondrial inner membrane protein prohibitin2(PHB2),both directly involved in mitochondrial autophagy.qRT-PCR results demonstrated an increased expression of hsa-miR-371a-5p and hsa-miR-2355-5p in sperm as motility declined.Conclusion:This study identified miRNAs with specific dysregulation in the sperm of asthenozoospermia patients and their target genes,offering new insights and a theoretical basis for further investigation into the mechanisms by which miRNAs regulate mitochondrial autophagic functions in low motility sperm.