FBXO31泛素化作用在早发性卵巢功能不全发病中的机制研究

Premature ovarian insufficiency (POI) is an ovarian disorder of multifactorial origin which imposes great challenges on women’s fertility and premature menopause. However, the etiology of POI remains undefined. Abnormality of granulosa cell apoptosis, inadequate primordial follicle pool, or accelerated depletion of oocytes and follicles may contribute to the pathogenesis of POI. Our previous study has showed that poor ovarian reserve was associated with decreased levels of miR-106a in serum and granulosa cells, and increased FBXO31 mRNA expression in granulosa cells, which was exactly regulated by miR-106a. FBXO31, which acts as E3 ligase, regulates the expression of various target proteins (for example, p53,MDM2) by ubiquitin-proteasome degradation. To better understand the pathogenesis of POI, in the present study, we plan to investigate the potential role of FBXO31 ubiquitination to p53 and the feedback regulation of p53 on miR-106a levels in granulosa cell apoptosis and follicle development and atresia in vitro (granulosa cell culture and follicle culture) and in vivo (POI mice model and FBXO31 knockout mice).

早发性卵巢功能不全(POI)导致的不孕、早发绝经等严重影响女性生殖健康，卵泡消耗和闭锁加速是其主要原因。卵泡闭锁实际是卵泡颗粒细胞及卵母细胞的凋亡。我们前期研究发现POI患者血清和颗粒细胞中miR-106a水平下降，颗粒细胞FBXO31mRNA升高，FBXO31表达受miR-106a调控。结合FBXO31的泛素化作用，本研究拟围绕“FBXO31泛素化作用为中心的miRNA-基因-蛋白调控异常可能是POI发病的重要原因”这一假设，应用原代颗粒细胞和卵泡培养体系，结合POI小鼠和FBXO31敲除小鼠模型，通过在体miRNA过表达、ChIP等技术研究FBXO31对p53、MDM2等蛋白泛素化降解和p53对miR-106a转录调节，阐释以FBXO31泛素化作用为中心的miR-106a/FBXO31/p53环路调节在POI发病中的作用机制，为POI病因学研究和疾病干预提供理论基础。

早发性卵巢功能不全(POI)病因不明，其导致的不孕、早发绝经严重威胁女性生殖健康，任何影响卵泡池大小和加速卵泡闭锁的因素都会影响卵巢储备。卵泡闭锁实际是卵泡颗粒细胞及卵母细胞的凋亡。POI的病因和发病机制尚未完全明确，本研究应用原代卵巢颗粒细胞培养、三维卵泡培养体系、POI小鼠模型等，在细胞层面和整体水平研究以FBXO31泛素化为中心的miR-106a/FBXO31/p53环路调节在POI发病中的机制，为POI病因学研究和疾病干预提供理论基础。.1）在POI患者的颗粒细胞中，FBXO31呈高表达状态，而miR-106a呈低表达状态，皮尔森相关分析中两者在RNA表达水平呈负相关，并验证了FBXO31是miR-106a靶基因。.2）FBXO31可引起人颗粒细胞凋亡增多，凋亡相关蛋白Bax、p53表达增多，Bcl-2表达降低。酵母双杂交实验显示FBXO31与MDM2可以直接结合。过表达FBXO31的颗粒细胞中出现细胞的p53含量增多（特别是细胞核定位的p53）、多个抗氧化酶表达降低、线粒体的Bax蛋白增多，引起细胞活性氧增多，凋亡比例增加。.3）应用动物模型研究FBXO31对卵巢功能的影响：FBXO31过表达可使得卵巢内分泌功能减退，表现在抗米勒管激素（AMH）水平下降；FBXO31可引起小鼠各级卵泡数量减少，卵泡闭锁增加；FBXO31可导致卵母细胞DNA损伤和质量下降，表现为卵母细胞的活性氧显著增加、线粒体呈现异常团块状的分布状态。