MiR-601/SIRT1在多囊卵巢综合征高雄激素发生中的作用机制研究

Polycystic ovary syndrome (PCOS), characterized by hyperandrogenism, is one of the most common reproductive endocrine diseases. MiR-601, tends to be sorted into exosome and locates in the 9q33.3, which is a genetic susceptibility area founded by PCOS genome-wide association study (GWAS) and widely replicated in different populations. We found that miR-601 was increased in PCOS patients and positively correlated with testosterone level. In PCOS patients, the excess androgen mainly come from ovary theca-interstitial cells. So we over-expressed mmu-miR-5135, which has the same seed sequence with hsa-miR-601, and found the androgen synthesis increased in the mouse theca-interstitial cells. The expression of steroid-related genes (Lhcgr and Cyp17a1) also increased. By bioinformatics prediction and MirTrap experiment, we found miR-601 can directly bind SIRT1 mRNA, and then inhibit its expression. In the past, there are reports about SIRT1’s effect on androgen synthesis. For example, after knockdown or conditionally knockout SIRT1 gene, the male mouse show increased serum testosterone level. But this phenomenon is not reported in females and lack of mechanism research. Besides, ovary SIRT1 expression is decreased in DHEA induced PCOS-like rat model. Above all, we speculate that miR-601 may regulate androgen synthesis through SIRT1-mediated signaling pathway in the ovary theca-interstitial cells. In this project we plan to up/down regulate miR-601/5135 and SIRT1 in the human/mouse ovary theca-interstitial cells, use miR-601/5135 transgenic mouse and SIRT1 conditional knockout mouse model to explore the role of miR-601/SIRT1 on androgen synthesis and its molecular regulatory mechanism. At the same time, we will combine our clinical resource with above results to explain the pathological mechanism of the miR-601 abnormal expression in the pathogenesis of PCOS with hyperandrogenism. This program will provide new clues for PCOS diagnosis and treatment.

多囊卵巢综合征（PCOS）是以卵巢膜间质细胞合成过多的雄激素为主要特点的生殖内分泌疾病。本课题从GWAS易感区域内选取miR-601检测发现其在PCOS患者中显著升高，且与雄激素存在正相关关系。小鼠膜间质细胞中过表达miR-5135（miR-601的同源物）后，发现雄激素合成增加，Lhcgr和Cyp17a1表达增强。经生物信息学预测及体外实验证实，miR-601可以直接结合SIRT1抑制其表达。以往报道PCOS模型鼠SIRT1表达降低，雄鼠敲减或条件性敲除SIRT1后雄激素升高，但在雌鼠中未见报道。因此我们推测miR-601通过SIRT1信号通路调节膜间质细胞雄激素的合成。本项目将利用体外细胞干扰、体内miRNA转基因和SIRT1条件性敲除雌鼠共同研究miR-601通过SIRT1直接或间接调控雄激素合成的分子机制，并结合临床信息，解释miR-601异常参与PCOS高雄激素发生的作用机理。

多囊卵巢综合征是以雄激素增高为核心的常见生殖内分泌疾病。本课题从遗传（GWAS易感位点）和表观遗传（miRNAs）结合的角度出发，选取在不同人群中经过广泛验证的易感区域内的miR-601作为研究对象，利用体内外实验（细胞与基因修饰小鼠）研究miR-601在PCOS高雄激素血症发生中作用与分子机制。在本项目的资助与支持下，我们发现miR-601在膜间质细胞（负责雄激素合成）中主要作用于FAM60A，进而调节雄激素合成关键基因CYP17A1等的转录表达；而在颗粒细胞（负责雄激素转化）中主要靶向调控SIRT1，进而影响雌孕激素合成相关基因的表达。SIRT1抑制剂尼克酰胺在体内外同样能剂量依赖性的调节雌孕激素合成。基于临床数据分析发现，miR-601在PCOS患者颗粒细胞中显著高表达，其靶基因SIRT1在PCOS患者颗粒细胞中显著低表达，且在PCOS严重高雄的患者中表达更低。即PCOS患者异常表达的miR-601/SIRT1通过影响卵巢雄激素合成与转化参与PCOS高雄激素的发生发展。该课题的实施为解析GWAS易感区域的miRNA在PCOS发病中的作用提供了新的理论依据，为从miRNA 途径改善PCOS高雄状态提供了新的靶点。