长非编码RNA NLC1-C及其相关蛋白Nucleolin在男性不育发生中的作用及其分子机制

Spermatogenic failure is one of the major reasons of male infertility. Non-coding RNAs, eg. microRNAs and long non-coding RNAs, have been recently emerged as key regulators of eukaryotic gene expression in the spermatogenesis. Our previous results indicated that long non-coding RNA NLC1-C was predominately expressed in both cytoplasm and nuclei of spermatogonia and primary spermatocytes in spermatogenesis, but cytoplasmic NLC1-C was significantly down-regulated and nuclear NLC1-C was increased in male infertility patients with mixed pattern of maturation arrest (MA). NLC1-C associated protein Nucleolin was mainly localized in spermatocytes, but in testicular samples of MA, the spermatocytes signal was excluded and a dislocated expression of Nucleolin was observed in spermatogonia. Our results demonstrated that NLC1-C was a key regulator in spermatogenesis failure. However, the role and the molecular mechanism of NLC1-C in MA need to be further investigated. Thus, our project is aimed to: 1) detect the expression of NLC1-C and Nucleolin in different types of non-obstructive azoospermia (NOA), including MA and hypospermatogenesis patients’ testicular biopsies; 2) investigate the molecular mechanism of NLC1-C and its associated protein Nucleolin in MA. To achieve this goal, I will use high throughput sequencing to identify and characterize genes involved in NLC1-C/Nucleolin pathway in spermatogenesis. Our study will provide new clues to personalized diagnosis and treatment for male infertility.

精子发生或成熟异常是男性不育的最主要原因。研究表明，非编码 RNA如miRNA、长非编码 RNA（lncRNA）在精子发生过程中起着重要作用。申请人前期发现，lncRNA NLC1-C在生精细胞成熟停滞患者（MA）睾丸组织中表达下调，且聚集在细胞核中；NLC1-C结合蛋白Nucleolin在MA中表达亦下降，其表达定位在正常睾丸组织的初级精母细胞核和MA的精原细胞核中。这些结果提示NLC1-C在男性不育中发挥重要作用。但NLC1-C作用的确切分子机制有待进一步探讨，因此，本项目拟开展：1）检测NLC1-C及Nucleolin蛋白在不同类型男性不育睾丸组织中表达情况；2）分别对NLC1-C/Nucleolin表达紊乱的病理组织和体外模拟的睾丸癌细胞进行高通量基因测序，探究因NLC1-C/Nucleolin蛋白表达异常导致MA的相关基因，并探讨其分子机制,为男性不育的诊治提供新的线索。

利用RNA FISH方法检测长非编码RNA NLC1-C表达在正常睾丸组织精原细胞和精母细胞的细胞质和细胞核，但在生精细胞成熟停滞（Maturationarrest，MA）睾丸组织中，细胞质中NLC1-C 表达降低，细胞核中表达聚集；与之结合的Nucleolin 蛋白主要表达在正常睾丸组织的初级精母细胞核中，精原细胞中表达非常少，但在MA 中，Nucleolin 在精原细胞核中表达升高，初级精母细胞中表达显著降低。利用核中过表达质粒pZW1-sno-NLC1-C 体外转染NT2 细胞，发现pZW1-sno-NLC1-C能够增加NLC1-C 与Nucleolin 结合。过表达pZW1-sno-NLC1-C 和GFP-Nucleolin 质粒，模拟MA 病理组织中NLC1-C 和Nucleolin 表达现象，利用高通量基因测序技术对收集的对照组和实验组以及正常的睾丸组织和NLC1-C/Nucleolin 表达异常的睾丸组织测序，发现118个共有的基因表达上调，131个基因表达下调。挑选5个可能与精子发生相关的基因在组织和细胞中验证与测序结果一致。提示NLC1-C和Nucleolin表达异常可能导致这5个基因表达失调产生生精细胞成熟停滞。为NOA患者的临床诊断和治疗提供理论依据。