Malat1介导的高AMH在PCOS发生发展中的作用及机制研究

Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders and a leading cause of female infertility and abnormal metabolism, and is significantly related to negative female health effects and increased social medical cost. The mechanism underlying the pathophysiology of PCOS remains to be illustrated. In our previous studies, we successfully constructed a differently expressed RNA transcriptome profile which includes long non–coding RNAs (lncRNA) and mRNAs in granulose cells (GCs) of PCOS patients compared with control via RNA sequence. We found that Malat1, which is involved in AMH signal transduction, is one of the differently expressed lncRNA in GCs of PCOS patient. Based on the previous results, we plan to further clarify Malat1-mediated effects and molecular mechanisms of AMH in the development of PCOS, by constructing mouse/cell model with Malat1 knock-in and cell model with Malat1 knock-down. The reproductive phenotype including reproductive endocrinology, oogenesis of the animal model and proliferation/apoptosis/steriogenesis of cell models will be analyzed to confirm whether dis-regulated Malat1 will impaire oogenesis and steroidogenesis similar with PCOS. A series of experiments by RNA immunoprecipitation , real-time PCR, Western-blot , siRNA transduction and chromatin immunoprecipitation will be employed to explore the effects and mechanisms of AMH in up-regulating Malat1, and to figure out the crucial steps, the key regulatory factors and the precise regulatory network of AMH and Malat1 in PCOS development. The expected results will provide valuable theoretical basis for early screening and interference of PCOS, which will contribute to improving female reproductive health, and reducing related health-care costs.

多囊卵巢综合征（PCOS）发病率高，可致生殖障碍及代谢异常，严重危害女性健康，增加医疗负担，其发病机制尚未阐明。本课题组前期通过转录组测序构建了PCOS患者颗粒细胞mRNA及lncRNA差异表达谱，发现与AMH信号通路密切相关的lncRNA Malat1与PCOS发病之间存在关联。本研究拟进一步采用Malat1基因敲入小鼠模型、Malat1过表达/敲减细胞模型，观察生殖内分泌/卵子发育表型、细胞增殖/凋亡、甾体激素调节关键分子，阐明Malat1表达异常致PCOS发病的效应。通过AMH与Malat1关联分析，RNA免疫共沉淀、染色质免疫共沉淀、荧光素酶报告基因、siRNA等技术探索AMH调节 Malat1表达、参与PCOS发病的关键步骤及调控因子，明确Malat1介导AMH在PCOS发生发展中的作用及精细调节网络，为PCOS的临床筛查与靶向干预提供理论基础，兼具重大科研价值和转化医学意义。

多囊卵巢综合征（polycystic ovary syndrome, PCOS）是一种复杂、异质性的内分泌代谢紊乱症候群，是育龄妇女无排卵性不孕最主要的原因，我国育龄女性中PCOS发病率高达5.6%，严重影响生殖健康。PCOS典型的临床表现有高雄激素血症、稀发排卵/无排卵和卵巢多囊样改变等。卵泡发育阻滞造成的持续性无排卵是导致PCOS患者生殖障碍的主要原因，研究PCOS患者排卵障碍的机制，有助于了解PCOS的病理生理机制，并有助于预防长期无排卵导致的严重并发症。目前，国内外均已有研究表明PCOS患者血清中抗苗勒管激素(anti-Müllerian hormone, AMH) 水平较正常女性明显增高，PCOS患者AMH水平的升高可能在卵泡发育中发挥重要作用。长链非编码RNA (Long non-coding RNA, lncRNA) 广泛参与细胞的定向分化、生长发育。而lncRNA是否参与AMH在卵泡发育中的作用尚不清楚。在本研究中，我们通过高通量测序分析了PCOS患者和非PCOS患者卵巢颗粒细胞(granulosa cells，GCs)中lncRNA的表达情况。结果显示，共有79个非编码转录本在PCOS患者的GCs中有不同表达，其中lncRNA MALAT1表达上调。进一步采用Real-time qPCR扩大PCOS患者颗粒细胞样本，结果表明MALAT1在PCOS患者的GCs中表达上调。此外，在体外利用siRNA敲低KGN细胞系中MALAT1的表达可以促进KGN细胞的增殖。这些数据表明MALAT1在PCOS的发生发展中具有一定的作用。同时，利用外源性AMH刺激KGN细胞后，MALAT1和磷酸化的SMAD 1/5 (Ser463/465)蛋白表达上调，表明AMH可能是MALAT1表达的调控因子。通过ROC曲线分析，我们发现MALAT1可以在一定程度上预测临床妊娠结局(面积:0.771,P=0.007, 95% CI:0.617-0.925，敏感性:57.1%，特异性:91.7%)。综上，我们揭示了lncRNA MALAT1在抑制卵泡颗粒细胞增殖中的作用，并可能与PCOS的妊娠结局相关。