Project 2: Role of small RNAs in male infertility

项目概述（见说明书）：微rna （miRNA）和小干扰rna （sirna）被认为是翻译后mRNA修饰的关键调控因子，能够同时影响多个基因的表达。在人类睾丸中已经发现了小rna，并有望在人类生殖的理解方面取得重大突破。miRNA测序数据（人类睾丸）对比分析显示，70%的人类miRNA在物种之间高度保守，因此相对容易在动物模型中研究候选miRNA的作用机制。目前的建议旨在使用多重深度测序来鉴定新的小rna，并使用qRT-PCR来鉴定在男性不育中起关键作用的mirna。细胞分离将用于丰富不同细胞的种群，从而提高检测精原细胞、精母细胞、间质细胞和支持细胞中特定mirna的灵敏度。可育男性睾丸、仅支持细胞综合征和成熟阻滞男性的表达谱将被评估。将分析正常男性和严重少精症（密度< 1ml /ml）男性的精子miRNA表达，以确定射精精子miRNA谱分析是否可用于诊断男性不育的遗传原因。随后的实验将集中在一个保守的和x连锁的miRNA与一组在生殖中重要的基因相互作用的子集。从可育和不育患者中获得的TruSeq mRNA谱将用于实验鉴定关键的miRNA:mRNA靶标。选定的目标将在转基因模型中进一步确认，作为U54中心合作努力的一部分。在拟议的资助期结束时，这项工作将确定并确认一组大约20-30个在男性不育中至关重要的miRNA:mRNA相互作用，验证它们在体外的靶特异性，描述它们的作用机制，并将它们分配到已知的调节途径。这将为体内模型的发展奠定坚实的基础。然后，这些模型可用于测试外源性mirna或其抑制剂的递送是否可以挽救不育表型。这项工作也将为开发基于rna的治疗不孕症和其他睾丸疾病的方法提供见解。

PROJECT SUMMARY (See instructions): Micro RNAs (miRNA) and small interfering RNAs (siRNAs) are considered key regulators of posttranslational mRNA modifications, capable of affecting the expression of multiple genes simultaneously . Small RNAs have been identified in human testis, and hold promise for identifying major breakthroughs in the understanding of human reproduction. Comparative analysis of sequencing of miRNA data (human testis) showed that 70 % of human miRNA is highly conserved between species, thus allowing for relatively easy study of the mechanism of action of candidate miRNA in animal models. The current proposal aims to use multiplexed deep sequencing to identify new small RNAs, and to employ qRT-PCR to identify miRNAs critical in male infertility. Cell isolation will be used to enrich the population of different cells, allowing increased sensitivity to detect specific miRNAs in spermatogonia, spermatocytes, Leydig cells, and Sertoli cells. Expression profiles from fertile male testes, men with Sertoli cell only syndrome and maturation arrest will be evaluated. Expression of sperm miRNA in normal men and men with severe oligospermia (density <1 mil/ml) will be analyzed to determine if the ejaculated sperm miRNA profiling may be useful in diagnosis of genetic reasons for male infertility. Subsequent experiments will focus on a subset of conserved and X-linked miRNA interacting with set of genes important in reproduction. TruSeq mRNA profiles obtained from fertile and infertile patients will be used to experimentaly identify critical miRNA:mRNA targets. Selected targets will be confirmed further in transgenic models as part of U54 center collaborative effort. At the end ofthe proposed funding period, this work will identify and confirm a set of approximately 20-30 miRNA:mRNA interactions that are critical in male infertility, verify their target specificity in vitro, delineate their mechanisms of action, and assign them to known regulatory pathways. This will lay a solid groundwork for the development of in vivo models. These models can then be used to test whether delivery of exogenous miRNAs, or their inhibitors, can rescue the infertile phenotype. This work will also provide insights for the development of RNA-based therapies for infertility and other testicular disorders.