长链非编码RNA GRIFIN-L在原发性卵巢功能不全发病中的作用及机制研究

Premature ovarian insufficiency (POI), defined as cessation of menstruation before the expected age of menopause, is one of the commonest reproductive endocrine disorders for women of child bearing age. The underlying explanation for POI remains to be elucidated in most cases. Therefore, it is necessary to explore the pathogenesis of POI from other viewpoints. The results of our previous research showed that long non-coding RNA GRIFIN-L and an important oocyte-derived growth and differentiation factor 9 (GDF9), which was a possible targets of GRIFIN-L, were down-regulated in the granulosa cells of patients with POI. Silence of GRIFIN-L influenced the proliferation of ovarian granulosa cells and the expression of GDF9 gene. It is presumed that dysfunction of GDF9, regulated by GRIFIN-L, participate in the development of POI. Based on the previous results, the present study focuses on the effect of GRIFIN-L on the biological behaviors of granulosa cells, follicular development and ovarian function, and furthermore, illustrates the regulation of the target genes expression by GRIFIN-L, aiming to clarify underlying mechanism of GRIFIN-L in development of POI. Discovering non-coding RNA related with ovarian function will reveal the epigenetic mechanism contributing to the etiology of POI. Our genetic and functional studies will be helpful for the prevention, intervention and treatment of patients with POI.

原发性卵巢功能不全（primary ovarian insufficiency，POI）指女性40岁前闭经，伴促性腺激素升高及雌激素缺乏。POI具有高度的遗传异质性，候选基因研究主要围绕蛋白编码区变异，但仅能解释约10%患者的发病原因。因此，研究非编码序列在POI发病中的作用是一重要方向。申请人前期发现长链非编码RNA GRIFIN-L及卵母细胞源性生长因子GDF9（Growth and Differentiation Factor 9）在POI患者颗粒细胞中显著下调，且GRIFIN-L参与调控颗粒细胞增殖及GDF9表达，提示GRIFIN-L可能通过GDF9相关通路影响颗粒细胞及卵母细胞功能。本项目拟在前期基础上，探讨GRIFIN-L对GDF9及相关通路的调控作用及对颗粒细胞及卵母细胞的影响，从表观遗传角度揭示非编码RNA参与POI的分子机理，对于POI的早期诊断、早期干预具有重要意义。

女性生育力是人类繁衍的珍稀资源，卵巢储备是生育力的决定者和体现者。卵巢储备是指卵巢皮质内原始卵泡生长发育成为可受精卵母细胞的能力。女性在40岁前卵巢储备耗竭即为原发性卵巢功能不全（primary ovarian insufficiency，POI）。2015年12月欧洲人类生殖与胚胎学会（ESHRE）将POI重新定义为女性40岁之前前丧失卵巢活性，表现为闭经或月经稀发，伴有促性腺激素升高和雌激素降低。诊断标准将FSH阈值由高于40IU/L降至>25IU/L，诊断阈值的下调势必将更多的女性纳入POI 的管理范畴。POI发病率1%-2%，在我国约累及500万育龄期女性。POI患者不仅表现为闭经、生育障碍，且由于低雌激素水平而表现出一系列围绝经期症状。POI严重影响了女性的长期身心健康。.研究非编码RNA在POI发病机制中的作用是目前重要方向之一。本项目重点关注长非编码RNAGRIFIN-L在POI发病中的作用及机制，明确了卵巢颗粒细胞中lncRNAGRIFIN-L表达情况与血清AMH、FSH水平的相关性。在体外实验中，证实了GRIFIN-L在KGN细胞系中的功能。利用RNApull-down将GRIFIN-L直接结合的蛋白进行液相色谱-质谱联用鉴定后发现，剪接因子hnRNPK是与GRIFIN-L直接结合的蛋白。同时探究了lncRNADDGC在POI中的作用与机制，利用转录组测序的方法检测了DDGC在颗粒细胞中潜在的生物学功能，发现沉默DDGC可以导致KGN和SVOG细胞DNA损伤修复功能障碍。DDGC通过吸附miRNA调控靶基因的作用机制。该项目为发现卵巢功能相关非编码RNA提供实验基础和理论依据，对POI防治具有重要意义。.在该项目资助下发表SCI论文20篇，在项目资助期间项目负责人获山东省自然科学一等奖。