miR-135b靶向Hippo信号通路在PCOS发生中的作用和机制研究

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women, which is characterized by abnormal folliculogenesis. Our previous studies have proven that the expression of miR-135b was significantly up-regulated in cumulus cells from PCOS patients (PCOS vs normal, FC=21.29). We further identified that LATS2, an important gene of Hippo signaling pathway, is one of the targets of miR-135b. Here, we suggest a hypothesis that miR-135b could inhibit Hippo signaling pathway by targeting LATS2, thus resulting in the simultaneous growth of multiple early antral follicles in the development of PCOS. To prove this hypothesis, we aimed to define the molecular roles of miR-135b-mediated regulation of Hippo signaling pathway in cell proliferation, ovarian function and follicle development in vitro (KGN cells) and in vivo (transgenic mice). Moreover, the interplay between miR-135b and Hippo signaling pathway in the development of PCOS will be explored by using the miR-135b antagomirs to inhibit the function of miR-135b in PCOS models in combination with granulosa cells -specific YAP knockout mice. Finally, the in vivo effects of miR-135b antagomirs to improve the function of ovary in PCOS models will be examined. In sum, our findings will elucidate the molecular mechanism underlying the development of PCOS with a novel therapeutic avenue for the treatment of PCOS.

多囊卵巢综合征是育龄妇女常见的内分泌紊乱疾病，主要特征是排卵障碍。本课题组前期工作发现miR-135b在PCOS患者颗粒细胞中显著高表达（FC=21.29），并证实Hippo信号通路的关键基因LATS2是miR-135b的靶基因。在此基础上，我们推测miR-135b是靶向调控了Hippo信号通路，从而导致了PCOS的发生。为了验证该假设，本课题将通过在KGN细胞系（体外）和转基因小鼠（体内）感染miR-135b慢病毒载体，确定miR-135b调控Hippo信号通路对细胞增殖、卵巢功能及卵泡发育的影响；并在卵巢颗粒细胞条件性敲除突变鼠（YAPcko）的PCOS模型中，通过miR-135b antagomirs抑制miR-135b的表达，阐明miR-135b对Hippo信号通路的调控作用以及在PCOS发生中的机制。研究结果不仅会揭示PCOS的发病机理，而且有望寻找到新的治疗PCOS的药物靶点。

多囊卵巢综合征是育龄妇女常见的内分泌紊乱疾病，主要特征是排卵障碍。本项目以“PCOS卵泡发育的调控机制”为核心，着眼于卵泡发育的微环境，重点研究了卵巢颗粒细胞差异基因和卵泡液中小分子物质对卵泡发育的调控机制。研究发现，（1）作为一种原发性卵巢疾病，PCOS卵巢颗粒细胞中miR-135b异常升高，靶向抑制了Hippo信号通路关键酶基因LATS2的表达，导致Hippo通路异常激活，颗粒细胞增殖加速，卵巢皮质加厚，雄激素升高，阐明了PCOS高雄激素血症发生及其对卵巢发育的影响机制；（2）非编码RNA在PCOS卵泡发育的过程中起重要作用，本项目证实PCOS颗粒细胞中的lncRNA表达异常，PWRN2靶向调控TMEN120B的表达，在PCOS脂代谢异常过程中起重要作用，初步揭示脂代谢异常引起的卵泡液微环境改变，是影响卵泡发育的重要原因。（3）PCOS卵泡液外泌体携带的高表达的circRNA（cicrLDLR）通过ceRNA的机制下调了芳香化酶基因（CYP19A1）基因的表达，阻碍了雄激素向雌激素的转化，导致PCOS高雄伴慢性炎症状态发生，严重影响了卵泡的发育。（4）从代谢组学角度，采用拉曼光谱技术，分析了PCOS卵泡液中特异的光谱谱带及其对应的代谢物。并通过构建机器学习模型的方法，分析了特异光谱特征对胚胎发育和妊娠结局的预测价值。综上，本项目从基因组学和代谢组学对PCOS卵泡发育的调控机制进行了系统深入的探讨，初步揭示了PCOS卵巢颗粒细胞基因（mRNA、miRNA、lncRNA）表达异常，导致雄激素升高和炎症因子的增加，使卵泡液呈现出高雄伴慢性炎症状态，影响了卵泡的发育，最终导致PCOS发生的调控机制，研究结果对于治疗PCOS的靶向小分子药物的研发具有重要的理论价值，同时研究中提出的“微量卵泡液代谢物光谱特征预测胚胎发育和妊娠结局的机器学习模型”具有重要的临床应用价值。