弱精症相关lncRNA ASAL1在人成熟精子中的功能与机制研究

Long non-coding RNAs (lncRNA) are novel regulatory molecules of the numerous physiological and pathological processes. However, lncRNA whether or not participate in the functional regulation of mature sperm and its abnormal expression whether or not associate with male infertility is completely not reported. The applicant’s study has proved that the presence of sperm-specific and intact lncRNA in mature human sperm, significantly different expression profiles of lncRNA between the normal sperm and asthenospermia sperm. ASAL1, a testes/sperm specific-expressed lncRNA, is 14.8-fold compared with the normal population, and its expression is related to sperm motility. We proposed that ASAL1 play an important role in human mature sperm function. The project intends to validate the relationship of ASAL1 with asthenospermia and explore the probability that lncRNA such as ASAL1whether could serve as molecular biomarkers of asthenospermia. We will address the roles of ASAL1 based on the fluorescently labeled and in vitro transcribed lncRNA by gene electroporation. Furthermore, hunting for target protein complexes of lncRNA using molecular biology technology illustrate the underlying mechanisms. The successful implementation of this project will firstly discover the key lncRNA regulated mature sperm, help to reveal the lncRNA-related regulatory mechanisms of mature sperm function, and provide a potential target molecule for the diagnosis and treatment of asthenospermia.

长链非编码RNA（lncRNA）是众多生理和病理过程的新型调节分子。然而，lncRNA是否参与成熟精子功能调控、其异常是否与男性不育相关尚未见报道。申请人最近的研究证明成熟精子中存在大量完整lncRNA，弱精症精子lncRNA的表达谱发生显著改变，睾丸/精子特异性lncRNA ASAL1在弱精症人群中的平均表达量是正常人群的14.8倍，且表达量与精子活力相关。我们推测ASAL1在人成熟精子功能调控中具有重要作用。本项目拟进一步：临床大样本验证ASAL1的表达与弱精症的相关性，探讨其作为弱精症分子标记物的可行性；通过电转染荧光标记的ASAL1研究其在精子中的功能；寻找ASAL1的关键互作蛋白，阐明ASAL1及互作复合体调控成熟精子功能的分子机制。本项目的成功实施将有助于率先发现调控人成熟精子功能的关键lncRNA，揭示成熟精子功能调控的lncRNA相关机制，为特定弱精症的诊疗提供潜在靶分子。

弱精子症是导致男性不育的重要病因，其病理机制远未阐明。lncRNA参与了各种生理病理活动的调节，但其是否参与精子功能调控及弱精症的发生尚不清楚。本项目基于前期研究基础假设lncRNA ASAL1在人成熟精子功能调控中具有重要作用，其异常表达可能与弱精症相关。为了验证上述科学假设，本项目主要从以下几个方面进行了系统深入的研究：系统表征成熟精子中lncRNA的表达特征；临床大样本验证ASAL1等睾丸/精子特异性lncRNA与弱精症之间的相关性，探讨其作为弱精症分子标记物的可行性；探究lncRNA ASAL1在成熟精子中的功能；寻找ASAL1的关键互作蛋白，阐明ASAL1及互作复合体调控成熟精子功能的分子机制。我们发现：（1）成熟精子中存在大量低丰度的、完整全长形式的、具有潜在功能的lncRNA；（2）弱精症精子lncRNA的表达存在显著异常、包括ASAL1在内的关键睾丸特异性lncRNA的表达异常与弱精症相关；（3） lncRNA可能主要通过影响RNA的运转、代谢等在精子形成过程中发挥作用；（4）人鼠保守的睾丸特异性lncRNA的缺失导致雄（男）性生育能力受损甚至不育；（5）正常生理条件下没有生理功能的lncRNA可能参与了不利胁迫下的雄（男）生育调控。这些重要研究首次系统的鉴定和表征了成熟精子中的lncRNA，提示lncRNA有望作为弱精症的分子标记物，关键睾丸特异性lncRNA对于精子发生与雄（男）性不育至关重要。本研究拓宽了我们对于lncRNA作用范围与调控机制的理解，有助于阐明少弱精症等相关男性不育疾病的病理机制。在本项目的资助下，共发表学术论文13篇，其中SCI论文11篇，申请发明专利5项。协助培养博士研究生1名，硕士研究生3名。总的来说，本项目达到了预期目标。