外泌体miR-132通过PTEN/PI3K/Akt通路调节卵泡发育及对卵巢早衰发生的作用研究

Follicular atresia or excessive exhaustion of primordial follicle is a key process in the pathogenesis of premature ovarian failure (POF), but the exact etiology and pathogenesis of POF are still not very clear. Recently, we have found that exosome miR-132 significantly increased in the serum of POF compared with control by miRNA chip data, and it could be absorbed by granular cells and induced the apoptosis of ovarian granular cells. In addition, bioinformatics analysis and previous studies showed that miR-132 could regulate PTEN/PI3K/Akt signaling pathway, which is one of the key pathogenetic pathways of POF. Therefore, we speculate that exosome miR-132 may participate in the follicular development and the pathogenesis of POF by the PTEN/PI3K/Akt pathway. The study is designed to investigate the effects and molecular mechanism of exosome miR-132 on the proliferation and apoptosis of granulosa cells, the follicular development and atresia by the culture of rat granulosa cells and early ovarian tissues in vitro and animal model in vivo, with PKH26 marker, the overexpression of miRNA and DOTAP liposome-mediated delivery technique. The study will help to understand the roles of exosome miR-132 in the follicular development and pathogenesis of premature ovarian failure through PTEN/PI3K/Akt pathway, and provide new ideas and potential targets for the prevention and treatment of premature ovarian failure.

卵泡闭锁或耗竭过快是卵巢早衰发病的重要环节，但其确切病因和发病机理尚不十分清楚。我们前期通过外泌体miRNA芯片数据发现：在卵巢早衰患者血清中外泌体miR-132明显升高，其可被颗粒细胞吞噬且诱导其凋亡。另外，通过生物信息学分析和已有研究显示，miR-132可调节PTEN/PI3K/Akt信号通路，而此通路为卵巢早衰关键致病通路之一。故我们推测外泌体miR-132可能通过PTEN/PI3K/Akt通路调节卵泡发育及参与卵巢早衰发生。本项目拟应用大鼠颗粒细胞、早期卵巢组织体外培养及在体动物模型，通过PKH26标志、miRNA过表达及DOTAP脂质体转染技术，研究外泌体miR-132对颗粒细胞增殖与凋亡、卵泡发育与闭锁的影响及分子机制。将有助于阐明外泌体miR-132如何通过PTEN/PI3K/Akt通路调节卵泡发育及在卵巢早衰发生中作用，为预防和治疗卵巢早衰的发生提供新思路及潜在的干预靶点。

卵泡闭锁或耗竭过快是卵巢早衰（POF）发病的重要环节，但其确切病因和发病机理尚不十分清楚。我们前期研究发现，在POF患者血清中外泌体miR-132明显升高，其可被颗粒细胞吞噬且诱导其凋亡，但有关miR-132在POF中的作用及其机制尚不清楚。本项目首先建立了POF小鼠模型，然后检测了POF小鼠血清和卵巢组织中miR-132的表达变化及其可能相关信号通路；在离体状态下，观察了外泌体miR-132-5p对颗粒细胞生长及相关信号通路的影响；通过卵巢囊注射，在体观察miR-132-5p激动剂对卵泡发育及相关信号通路的影响。研究结果显示，在环磷酰胺诱导的POF模型中，血清和卵巢组织中的miR-132-5p的表达均明显增加，而miR-132-3p在低剂时变化不大。通过TargetScan和miRWalk数据库预测显示，miR-132-5p靶基因与铁死亡相关通路显著富集，如PI3K-Akt信号通路。另外，在POF模型中，卵巢组织的铁含量和脂质过氧化物也均明显增加。铁死亡相关分子Slc7a11、Gclc、Gpx-4基因和蛋白的表达水平均明显下降。Pten蛋白表达明显上升，磷酸化Akt表达下降。离体状态下，外泌体miR-132-5p抑制颗粒细胞生长，诱导颗粒细胞凋亡，铁死亡相关分子Slc7a11、Gclc、Gpx-4其因和蛋白的表达水平均明显下降。Pten蛋白表达上升，磷酸化Akt表达下降。卵巢囊注射miR-132-5p激动剂后，卵泡闭锁明显增加，原始卵泡和次级卵泡明显减少。相关信号分子表达与其对颗粒细胞处理表达变化一致。此研究有助于阐明外泌体miR-132-5p在POF中作用及其相关的信号通路，为预防和治疗卵巢早衰的发生提供新思路及潜在的干预靶点。项目完成时共发表相关的研究论文8篇，做特邀会议报告3次，指导研究生4人和青年教师2人。