长链非编码RNA对卵巢功能的调控作用及在多囊卵巢综合征中的作用和机制研究

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in women of reproductive age, whose etiology is still unknown. It is reported that long non-coding RNAs (lncRNAs) play important roles in human diseases, development and stem cells, however, its roles remains unclear in reproductive medicine, including the regulatory function involved in ovary and the mechanism of polycystic ovary syndrome. Our previous study showed that a total of 862 lncRNA transcripts were dysregulated in mural granulose cells from women with PCOS, compared with those from women with normal ovarian reserve. The expressions of the 4 chosen lncRNAs were found to be consistent with the result of microarray, and they both have a positive correlation with the number of retrieved oocytes, suggesting that the dysregulation of lncRNAs may affect the physiological function of granulosa cells and follicular development. In addition, we found that the expression level of lncRNA NEAT1 was significantly decreased in women with advanced maternal age. Previous animal studies showed that NEAT1 deletion might cause luteal phase deficiency and lead to infertility, suggesting that lncRNA may be involved in the steroid metabolism. We aim to explore the regulatory effect of lncRNA on the mechanism of polycystic ovary syndrome by using granulosa cell model and animal model, and it would provide prevention or treatment targets for female infertility associated with PCOS and improve their outcomes in assisted reproductive technology (ART).

多囊卵巢综合征（PCOS）是排卵障碍性不孕的最常见病因，发病机制不明。长链非编码RNA（lncRNA）在人类疾病、发育、干细胞等领域发挥重要调控作用，但在生殖领域的作用鲜有报道，其在卵巢功能调控及PCOS发病中的作用亦不明确。申请人前期通过lncRNA微阵列基因芯片检测发现：在PCOS患者卵泡液颗粒细胞中有862条lncRNA的表达水平出现显著变化；初步筛选4条lncRNA进行检测，发现其表达趋势与芯片结果相符，且表达水平与患者的取卵数呈正相关关系，提示lncRNA表达失调可能影响颗粒细胞功能进而影响卵泡发育。我们另一研究发现，lncRNA NEAT1在高龄不孕女性中的表达水平显著下调，而此前有动物研究表明，NEAT1缺失引起黄体功能缺陷而导致不孕，提示lncRNA可能参与调控卵巢性激素合成与代谢功能。本项目拟通过体内外模型，探索差异lncRNA在PCOS中的调控作用和机制。

多囊卵巢综合征（PCOS）是排卵障碍性不孕的最常见病因，发病机制不明。长链非编码RNA（lncRNA）在人类疾病、发育、干细胞等领域发挥重要调控作用，但在生殖领域的作用鲜有报道，其在卵巢功能调控及PCOS发病中的作用亦不明确。申请人前期通过lncRNA微阵列基因芯片检测发现：在PCOS患者卵泡液颗粒细胞中有862条lncRNA的表达水平出现显著变化；初步筛选4条lncRNA ENST00000510619、ENST00000556578、HCG26和NR_028308进行检测，发现其表达趋势与芯片结果相符，且表达水平与患者的取卵数呈正相关关系。进一步选取HCG26进行细胞体内外研究，结果显示多囊卵巢综合征患者HCG26水平上调，并与窦状卵泡数相关。在KGN细胞中沉默HCG26能抑制细胞增殖和细胞周期进程、增加芳香化酶基因表达和雌二醇的产生。因此，HCG26可能通过影响颗粒细胞的增殖和激素分泌功能参与PCOS的发病。此外，通过对芯片结果的分析，并结合文献，我们同时筛选了lncRNA TUG1和非编码RNA—miR-29a进行研究：结果显示TUG1在PCOS患者卵巢颗粒细胞中表达上调，并与窦状卵泡数相关，制作受试者工作曲线（ROC曲线）显示TUG1在PCOS患者与对照组之间存在显著差异，沉默TUG1能抑制颗粒细胞增殖、促进MAPKs通路依赖的凋亡和p21依赖的自噬，但可能不影响细胞周期进展。沉默TUG1能增加芳香化酶表达和雌二醇生物合成。我们的结果表明，TUG1在PCOS 颗粒细胞中的表达增加可能促进了卵泡的过度激活和生长，可能破坏了优势卵泡的选择；PCOS患者颗粒细胞中MiR-29a显著下调，并与窦状卵泡数相关。ROC曲线显示miR-29a在PCOS患者与对照组之间存在显著差异。在KGN和COV434颗粒细胞系中过表达miR-29a能抑制细胞增殖、抑制细胞周期进展、降低芳香化酶表达和雌二醇生成。表明miR-29a可能通过影响颗粒细胞增殖和类固醇形成过程参与了PCOS的发病。我们的研究报道了PCOS患者和健康女性颗粒细胞中的lncRNA谱，并通过我们的研究提示异常的lncRNA和miRNA可能在颗粒细胞增殖和类固醇生成中发挥重要作用，为研究PCOS的发病机制提供了思路。