摘 要: MicroRNA (miRNA)是一段长约为22 nt的内源性非编码单链RNA, 研究表明miRNA对正常精子发生和雄性发育必不可少。为了探究miR-181b-5p在雄性生殖细胞发育中的功能以及寻找其靶基因, 首先采用流式细胞术、噻唑蓝检测以及划痕实验分析了miR-181b-5p在小鼠GC-1spg精原细胞系中的生物学效应, 随后利用Targetscan和GO数据库等生物信息学软件预测发现生殖核受体Nr6a1可能为其靶基因, 进而通过双荧光素酶报告基因实验以及qPCR证实了miR-181b-5p与Nr6a1的靶向识别关系, 最后采用细胞功能回补实验明确了miR-181b-5p通过靶向调控Nr6a1来抑制GC-1spg细胞的增殖和迁移。结果表明miR-181b-5p通过其靶基因Nr6a1在精子发生中起到负调控的作用。

Abstract: miRNA, approximately 22 nts in length, is an endogenous and non-coding single-stranded RNA. Some researches indicated that miRNA is essential to normal spermatogenesis and male development. In order to find out the impact and target gene of miR-181b-5p in germ cell development, flow cytomety, MTT and scratch assays were used to analyze the biological effects of miR-181b-5p on mouse GC-1spg cells. Then Nr6a1 was predicted as one of target genes of miR-181b-5p by bioinformatics software such as Targetscan and GO database. Subsequently, the target recognition between miR-181b-5p and Nr6a1 was verified through dual luciferase reporter gene assay and qPCR. Lastly, by functional complementation assay, it proved that miR-181b-5p could inhibit proliferation and migration of GC-1spg cells through targeting Nr6a1. These results suggest that miR-181b-5p plays a negative role in regulation of spermatogenesis by targeting Nr6a1.