Project 2: Role of small RNAs in male infertility

项目摘要(见说明)：microRNAs(MiRNA)和小干扰RNAs(SiRNAs)被认为是翻译后mRNA修饰的关键调节因子，能够同时影响多个基因的表达。已经在人类睾丸中发现了小RNA，并有望在理解人类生殖方面取得重大突破。对miRNA数据(人睾丸)的测序比较分析表明，70%的人miRNA在物种之间高度保守，从而允许在动物模型中相对容易地研究候选miRNA的作用机制。目前的建议旨在使用多重深度测序来识别新的小RNA，并使用qRT-PCR来识别在男性不育中起关键作用的miRNAs。细胞分离将用于丰富不同细胞的数量，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miRNAs的灵敏度。我们将评估有生育能力的男性睾丸、患有仅支持细胞综合征和成熟停滞的男性的表达谱。将分析正常男性和严重少精子症(密度为1mil/ml)男性精子miRNA的表达，以确定射出的精子miRNA图谱是否有助于诊断男性不育的遗传原因。随后的实验将重点放在保守的和X连锁的miRNA子集与一组在生殖中重要的基因相互作用上。从可生育和不育患者中获得的TruSeq信使核糖核酸图谱将被用于实验性地识别关键的miRNA：信使核糖核酸靶标。作为U54中心合作努力的一部分，选定的靶点将在转基因模型中得到进一步确认。在拟议的资助期结束时，这项工作将识别和确认一组大约20-30个在男性不育中至关重要的miRNA：mRNA相互作用，在体外验证它们的靶标特异性，描述它们的作用机制，并将它们分配到已知的调控途径。这将为体内模型的开发奠定坚实的基础。然后，这些模型可以用来测试外源miRNAs或其抑制剂是否可以拯救不育的表型。这项工作还将为基于RNA的不育症和其他睾丸疾病治疗方法的开发提供见解。

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.