miR125b介导AR剪接变异体抑制PCOS卵巢颗粒细胞凋亡的机制研究

The important role of androgen receptor (AR) in the pathogenesis of polycystic ovary syndrome (PCOS) has been confirmed by AR knockout animal model. Our previous studies have found that AR alternative splicing variants(ASVs) happened in granulosa cells (GC) of PCOS patients, which were strongly associated with hyperandrogenism and abnormalities in folliculogenesis of PCOS. Furthermore, we found GCs expressing AR ASVs show suppressed apoptosis, which may be regulated by miR125b. In this project, we plan to detect AR ASVs and miR125b expression in GCs of PCOS patients by Taqman PCR and detect apoptosis protein expression by Western blot. The relationship between AR ASVs and miR125b will then be explored by overexpression of AR ASVs and overexpression or knock down of miR125b in primary GC. Apoptotic proportion and expression of apoptosis protein will be delected by flow cytometry and Western blot, respectively. The protein interacting with AR ASVs will be is obtained by Co-IP and interpretated by LC - MS technology. MiR125b promoter binding protein will be obtained by DNA - pull down and followed with LC-MS technology. This project will reveal the new epigenetic mechanisms of follicular developmental disorder in PCOS patients.

雄激素受体（AR）在多囊卵巢综合征（PCOS）发病机制中的重要地位已得到动物模型的证实。 本课题组前期发现以高雄和排卵障碍为特征的PCOS患者颗粒细胞（GC）中存在AR剪接变异体这类表观遗传异常. 前期结果显示AR剪接变异体存在颗粒细胞中凋亡下降，并发现由靶基因miR125b调控。本项目拟通过定量PCR等技术明确PCOS患者GC中各型AR剪接变体以及miR125b的表达丰度，通过Western blot检测凋亡情况。在原代培养的正常颗粒细胞中过表达AR剪接变异体，敲低（或过表达）miR125b明确两者调控关系，通过流式细胞计数及检测凋亡相关蛋白表达量检测细胞凋亡情况。通过Co-IP及LC-MS技术明确与AR剪接变异体相互作用蛋白种类，通过DNA-pull down技术，获得miR125b启动子结合蛋白种类。 揭示miR125介导AR剪接变异体导致PCOS患者卵泡发育障碍的新的表观遗传机制。

雄激素受体（AR）在多囊卵巢综合征（PCOS）发病机制中的重要地位已得到动物模型的证实。 本课题组前期发现以高雄和排卵障碍为特征的PCOS患者颗粒细胞（GC）中存在AR剪接变异体这类表观遗传异常. 前期结果显示AR剪接变异体存在颗粒细胞中凋亡下降，并发现由靶基因miR125b调控。后期通过我们的研究发现，Ins型AR和Del型AR剪接变异体均存在入核障碍。其中Ins型AR剪接变异体增加miR125b表达并不依赖雄激素，从而抑制卵巢颗粒细胞的凋亡。通过联合CoIP-MS及DNA Pull down-MS技术，发现腺嘌呤核苷转移酶2（ANT2）蛋白与Ins型AR剪接变异体相互作用，同时也能结合到miRNA125b启动子上。本研究中miRNA125b、上游ANT2及雄激素剪接变异体相互调控机制，加深对PCOS 发病机理的认识，为治疗提供了可能的靶点。