circPVT1调控miR-125b/PaK3在PCOS高雄激素血症中作用机制研究

Hyperandrogenism is a major biochemical characteristic of PCOS, which is related to follicular dysplasia and metabolic disorder, and its pathogenesis is still unclear. There are few reports on the regulation of follicular development and hormone secretion by circular RNA (circRNA) and microRNA (miRNA). Based on our preliminary results, the negative regulation of miR-125b in Pak3 affects the expression of steroid hormone synthetase, which promotes the secretion of androgen. Bioinformatics and previous publications suggest that circPVT1 could be a sponge to adsorb miR-125b and reduce its activity. circPVT1 is highly expressed in follicular fluid of HA-PCOS patients and promotes testosterone secretion by Pak3 in follicles. Therefore, the hypothesis that "circPVT1 regulates miR-125b/PaK3 to play a role in the occurrence of hyperandrogenism inPCOS " is proposed.This study starts from investgating theca cell steroidogenesis enzymes and intends to using the overexpression/ silencing/ salvaging strategy, luciferase activity analysis and fluorescence probe techniqueswere in theca cells, follicles, animal models and clinical samples of four levels to explore this hypothesis. Our results will be helpful to reveal the molecular mechanism of PCOS hyperandrogenism and provide new clues to discover new biomarkers and prevention measures in PCOS.

高雄激素血症是PCOS主要生化特征，与卵泡发育障碍及代谢紊乱发生相关，其发病机制尚不清楚。环状RNA（circRNA）和微小RNA（miRNA）与卵泡发育及激素分泌调控的研究报道甚少。前期发现miR-125b负向调控Pak3影响甾体激素合成酶的表达促进雄激素的分泌。生物信息学和文献提示，circPVT1可作为海绵吸附miR-125b降低其活性；其在PCOS高雄激素血症患者卵泡液中高表达，在卵泡中调控Pak3促进睾酮分泌。因此提出“circPVT1调控miR-125b/PaK3，在PCOS高雄激素血症发生中发挥作用”的假说。本研究拟：以卵泡膜细胞甾体激素生成酶为切入点，利用过表达、沉默及挽救实验策略、荧光素酶活性分析及荧光探针等研究手段，在卵泡膜细胞、卵泡、动物模型和临床标本四个层次探讨这一科学假说，以揭示PCOS高雄激素血症发生的分子机制，为寻找早期生物标志及发现新的防治措施提供科学依据。

高雄激素血症是多囊卵巢综合征(polycystic ovary syndrome, PCOS)的主要特征之一，与卵泡发育障碍及代谢紊乱发生相关，其发病机制尚不清楚。环状RNA（circRNA）和微小RNA（miRNA）与卵泡发育及激素分泌调控的研究报道甚少。miR-125b-5p的异常表达已在多种疾病中被证实，但miR-125b-5p是否与窦前卵泡的异常类固醇生成相关仍不清楚。我们检测了PCOS患者临床血清样本中的类固醇激素浓度和miR-125b-5p表达水平，并利用小鼠窦前卵泡培养模型和来曲唑诱导的PCOS小鼠模型，研究miR-125b-5p调控雄激素和雌激素分泌的机制。我们的研究发现miR-125b负向调控Pak3影响甾体激素合成酶的表达促进雄激素的分泌。此外，抑制miR-125b-5p促进了小鼠窦前卵泡中ERK1/2的激活，而抑制Pak3则减弱了这种激活作用。这些结果在来曲唑诱导的PCOS小鼠卵巢中可以重现。值得注意的是，抑制PAK3可拮抗miR-125b-5p siRNA促进雄激素合成相关酶表达和睾酮分泌的作用。黄体生成素(LH)可以抑制miR-125b-5p表达，刺激Pak3表达。PCOS患者高血清LH浓度可抑制miR-125b-5p的表达，进而上调Pak3的表达，激活ERK1/2信号通路，从而刺激HA-PCOS患者雄激素合成相关酶的表达和睾酮分泌，揭示了PCOS高雄激素血症发生的部分分子机制..此外，高通量测序和RT-qPCR结果提示 circEpha5在PCOS小鼠卵巢及PCOS患者血清中表达异常增高。FISH结果提示 circEpha5分布在卵泡膜细胞、卵巢间质细胞及原始卵泡的外周颗粒细胞的细胞质中。双荧光素酶报告基因实验发现circEpha5可以靶向结合miR-758-5p。功能试验表明，在窦前卵泡水平过表达circEpha5后miR-758-5p表达降低，CYP-17A1表达上升，窦前卵泡培养液中的游离睾酮含量增多；在窦前卵泡过表达miR-758-5p后circEpha5表达降低，CYP-17A1的表达降低，培养液中的睾酮水平减少。提示CircEpha5可海绵吸附miR-758-5p调控CYP-17A1表达，从而促进窦前卵泡水平雄激素的合成分泌，可能参与PCOS高雄激素血症的发生发展。.以上研究为临床发现合防治PCOS提供了新方向。