miR-200a-5p调节卵泡形成的分子机理研究

In mammals, the primordial follicle pool, providing all oocytes available to a female throughout her reproductive life is essential for female fertility. For human being, dys-regulation of primordial follicle assembly results in female reproductive diseases, such as premature ovarian insufficiency (POI) and infertility. As a well-understood post-transcriptional regulator, miRNAs have been studied in reproduction biology. However, the contribution of individual miRNAs to primordial follicle assembly remains largely unknown. Our previous work showed that the expression of miR-200a-5p decreased significantly during primordial follicle assembly, while overexpression of miR-200a-5p inhibited primordial follicle assembly. Its predicted target -- Cdh2 had a function role in primordial follicle assembly, but the mechanism for the regulation of Cdh2 expression is unknown. Therefore, by means of fetal mouse ovary in vitro culture system and conditional knockout mouse model, the present study will explain how miR-200a-5p regulates primordial follicle assembly through Cdh2, search for the upstream regulation factor of miR-200-a-5p, and uncover the mechanisms underline the diseases caused by abnormal primordil follicle assembly, which may finally bring new insights for researches on the pathogenesis of infertility as well as new tagets for its therapy, and further our understanding on molecular mechanisms of primordial follicle assembly.

哺乳动物原始卵泡库的大小决定了雌性生育期的长短，人类原始卵泡形成异常会导致原发性卵巢功能不全和不孕不育等疾病。作为广泛被研究的转录后调控因子，miRNA在生殖生物学中发挥着重要作用，但在卵泡形成过程中的调控作用及机制仍知之甚少。我们发现miR-200a-5p在原始卵泡形成过程中，表达明显下调，过表达可显著抑制原始卵泡形成，且其预测的靶基因Cdh2已被报道参与了原始卵泡形成，但Cdh2的表达调控机制尚不明确。因此，本项目拟利用胎鼠卵巢三维培养体系，结合Cdh2条件性敲除小鼠模型，对miR-200a-5p上游表达调控因子进行寻找验证，探讨miR-200a-5p如何作用于靶基因Cdh2，进而调控原始卵泡形成，揭示miR-200a-5p调控原始卵泡形成的分子机制，以期为卵泡形成异常导致的相关疾病诊治提供分子标记和靶点，为原始卵泡形成的分子调控机制提供新思维、增添新内容。

哺乳动物中，卵巢原始卵泡库的大小决定了雌性在整个生育周期中用于生殖的卵母细胞数。因此，原始卵泡库的形成对于雌性哺乳动物的生殖至关重要。在人类，这一过程的失调会导致多种生殖疾病，如原发性卵巢功能不全和不孕。越来越多的研究表明，原始卵泡形成受到了严格的转录和转录后水平的调控。作为广泛被研究的转录后调控因子，microRNA（miRNA）在不同的细胞生物学过程（包括生殖）中都发挥着重要作用。miRNA成熟过程所必需的核酸内切酶III——Dicer的缺失，会导致雌性小鼠卵泡异常和不育。但是，在原始卵泡形成过程中，miRNA的生物学功能仍然知之甚少。为确定哪些miRNA参与原始卵泡形成的调控，我们利用miRNA microarray检测在原始卵泡形成前及形成时的小鼠卵巢中差异表达的miRNA，发现18条在原始卵泡形成过程中差异表达的miRNA。我们对其中一条miRNA，miR-200a-5p进行了深入研究。miR-200a-5p通过抑制Cdh2的mRNA和蛋白的表达调控卵巢细胞之间的粘附与通讯，进而抑制原始卵泡的形成。我们发现，miR-200a-5p不但调控Cdh2的表达，其自身也受到雌激素的调控。利用条件性敲除小鼠模型，我们对卵巢体细胞和卵母细胞中的Cdh2的功能进行了研究，结果发现卵巢体细胞缺失Cdh2之后会抑制原始卵泡形成，而Cdh2在卵母细胞中的缺失并没有影响原始卵泡的形成，表明雌激素通过卵巢体细胞中miR-200a-5p与其靶基因Cdh2来抑制原始卵泡形成。我们的研究结果，不仅系统地证明了miRNA可参与卵泡形成，也为卵泡形成的分子调控机制增添了新内容。