miR-181a和miR-193a-3p协同调控精子活力参与男性不育发生的分子机制研究

The annual incidence of male infertility increases gradually, however, its molecular mechanism has not been reveled yet. Our previous studies have found out that the expression levels of miR-181a and miR-193a-3p were significantly upregulated in seminal plasma and mature sperm from patients with asthenospermia. A recent study applying bioinformatics, as well as the luciferase reporter assay have suggested that miR-181a and miR-193a-3p are able to regulate the expression of two sperm motility related proteins, PLAU and TNP1. Therefore, miR-181a and miR-193a-3p might be crucial and closely relate to the occurrence and development of male infertility by carrying out co-regulatory effect on PLAU and TNP1. In this study, the co-regulatory effects of miR-181a and miR-193a-3 to either PLAU or TNP1 and their detailed molecular mechanisms were investigated by applying sequential biological methods, which included real-time quantitative PCR, Western blot, immunofluorescence, lentiviral transfection, and RNA interference. And the experimental samples involved not only clinical specimens, but also cell lines and animals. In particular, whether the systematic regulatory network is correlated with the molecular mechanism of sperm motility and male infertility was overlooked. In addition, the present results will help us to better understand the mechanisms involved in sperm motility and provide a potential molecular therapy targets for male infertility.

男性不育发病率逐年上升，但发病机制尚不清楚。本课题组前期研究发现，弱精症患者精浆和精子中miR-181a、miR-193a-3p水平与正常对照相比显著上调；生物信息学预测及荧光素酶报告实验证实二者可同时调控精子活力相关蛋白PLAU和TNP-1表达。据此提出假说：miR-181a和miR-193a-3p协同调控PLAU、TNP1表达参与精子活力调节，与男性不育发生发展密切相关。为验证假说，本项目拟采用实时荧光定量PCR、Western blot、免疫荧光、脂质体及慢病毒转染、RNA干扰等技术，通过一系列临床标本、细胞和动物实验，明确miR-181a和miR-193a-3p对PLAU和TNP-1的协同调控作用，证实该调控网络与精子活力相关，阐明miRNA与靶基因构成的调控网络参与男性不育发生的分子机制，预期研究结果对男性不育发病机制和分子治疗具有重大理论和临床价值。

男性不育发病率逐年上升，但发病机制尚不完全清楚。微小核糖核酸（miRNA）是一类参与基因转录后水平调控的非编码小分子RNA，与睾丸发育、精子发生等生理过程密切相关，其表达异常可参与男性不育发生发展。人精浆中也存在多种稳定、丰富表达的miRNA，可为男性不育辅助诊断及发病机制研究提供新的思路和方向。本项目首先通过收集批量男性不育患者精浆、精子标本，运用qRT-PCR技术验证和发现精子活力低下男性不育患者精浆和精子中miR-181a和miR-193a-3p表达显著上调，与精子活力呈明显负相关；生物信息学分析、荧光素酶报告实验和western blot实验证实二者可同时调控精子活力相关蛋白PLAU表达；同时，PLAU蛋白在弱精症患者精子中水平明显降低，与miR-181a和miR-193a-3p表达变化相反；体外细胞实验结果显示，miR-181a和miR-193a-3p可通过调控PLAU的表达抑制睾丸支持细胞增殖；进一步的体内动物实验证实，环磷酰胺诱导的精子活力低下小鼠模型睾丸组织、精子中miR-181a和miR-193a-3p水平 也显著升高，而PLAU蛋白表达显著降低，两者的表达变化呈现明显负相关，与临床标本中变化完全一致，结合弱精症小鼠慢病毒过表达或抑制miR-181a/miR-193a-3p治疗模型，本项目研究结果表明：弱精症患者、小鼠精浆、精子和睾丸组织中miR-181a和miR-193a-3p表达明显上调，二者可共同调控PLAU表达参与精子活力调节，与男性不育发生发展密切相关，miR-181a/miR-193a-3p/PLAU调控通路可为精子活力异常发病机制和分子治疗提供了新的途径和理论依据。