猪miR-762通过RNF4-AR信号通路介导而参与调控精子发生的机制研究

miRNA是真核细胞中一大类参与基因转录后调控的非编码小RNA，在动物繁殖等过程中起重要作用。在国家自然科学基金项目（30700571）资助下，通过哺乳动物miRNA芯片及同源比较法鉴定出一个猪新miRNA—miR-762，且在大白猪性成熟前后睾丸组织差异表达，并通过双荧光报告系统对其靶基因RNF4进行了初步验证。然而猪miR-762在精子发生中的作用及相关机制尚不清楚。有鉴于此，本项目拟通过抑制表达、超表达等确定猪miR-762靶基因RNF4及靶位点，并在细胞水平上研究猪RNF4基因功能；综合利用免疫荧光、免疫共沉淀、ChIP、EMSA，鉴定RNF4的互作蛋白AR及其他调控因子；最后通过miR-762睾丸活体注射和睾丸条件性敲除小鼠模型，从活体水平较系统地研究miR-762通过RNF4-AR信号通路介导而参与精子发生的机制和调控网络。本项目研究为猪精子性状的遗传控制提供理论基础和新见解。

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that function as post-transcriptional regulators of target genes in eukaryotes, which play crucial regulatory roles in several life processes including animal reproduction. Supported by the previous NSFC grant (30700571), ssc-miR-762 was a novel porcine miRNA which was identified by a custom-made mammalian miRNA microarray and homology search approach. Moreover, miR-762 was differentially expressed in sexually immature and mature porcine testes from Large White boars, and RNF4 gene was identified as one target of miR-762 by dual-luciferase reporter system. However, it still remains unknown on miR-762 roles and the relative genetic mechanism of miR-762 in regulating spermatogenesis. In this proposal, we will validate RNF4 gene as one target of miR-762, and study the effect of porcine RNF4 on spermatogenesis through suppressed-expression and over-expression in vitro; identify RNF4-AR interaction and other RNF4 interactive factors by Co-IP, immune fluorescence method, ChIP and EMSA; then investigate the effect of miR-762 on spermatogenesis in vivo via miR-762 injection into testicular tissue and testicular -specific miR-762 knockout mice model. We expect to systematically investigate the essential role of miR-762 in spermatogenesis via RNF4-AR signaling pathway and to establish the miR-762 related genetic network in vitro and in vivo. This proposal will provide a basic foundation and a new insight into the genetic regulation of boar sperm traits.