子宫内膜异位症中芳香化酶DNA甲基化的调控机制

Endometriosis is an estrogen-dependent disease. Aromatase catalyzes the final step of estrogen production. Aromatase is undetectable in eutopic endometrial stromal cells, whereas is highly expressed in the stromal cells in endometriosis, which is equal to 2659-fold increase in the former. The Steroidogenic Factor-1 (SF-1) is a transcriptional factor essential for activation of aromatase gene. It is the first time that we reported that methylation of the promoter and intron CpG islands of SF-1 gene regulated the SF-1 expression in endometriosis and endometrium. IF=6.5, 2007; IF=2.6, 2011. One of these two articles was cited 35 times in 5 years. And I presented these two articles in 62th meeting of American Society for Repoductive Medicine in 2007 and 11th world congress on endometriosis in 2011, one was awarded "In Training Award"，and another was awarded "Rodolphe Maheux Travel Award" . SF-1 protein could bind to the promoter region of aromatase. Here, we identified a classical CpG island at the promoter region of the aromatase gene (the CpG island is about 1kb). Up to now no report of this CpG island with the aromatase gene regulation was found. We will investigate the methylation status of these CpG island in endometrial and endometriotic stromal cells by primary cell culture, bisulfite modification, PCR amplifications, clones, sequencing analysis, and determine this mechanism that aromatase gene may be down-regulated by DNA methylation, by plasmid construction, luciferase reporter gene assay, chromatin immunoprecipitation assay, and electrophoretic mobility shift assay. Also we will check methylation may hinder the binding of SF-1 with aromatase regulation region, and the regulation of aromatase expression, by cloning different gene regions in different lengths with different numbers of CpG, and in vitro methylation. Finally, we aimed to find the relationship among SF-1\aromatase\methylation\endometriosis.

子宫内膜异位症为雌激素依赖性疾病。芳香化酶是雌激素合成的关键酶，在异位病灶的间质细胞中高表达，在在位内膜的间质细胞中无表达，差异达2659倍。以往工作及上一基金期间我们首报了SF-1基因(芳香化酶的启动激活子)CpG岛的甲基化对SF-1有调节作用，发表2篇SCI论著，近5年单篇最高他引35次，获国际会奖两次。作为SF-1蛋白的结合部位，芳香化酶基因5'端有长1kb的CpG岛，是甲基化调节重要区域，但未见报道。我们将运用原代细胞培养、甲基化特异PCR、克隆、测序等手段，研究此CpG岛在异位和在位内膜中甲基化程度的差异；构建质粒、荧光素酶、染色质免疫沉淀、电泳迁移率等，探讨甲基化对芳香化酶功能的降调作用；克隆不同长度、含有不同CpG位点数，以及甲基化定点改变，探讨甲基化阻碍SF-1与芳香化酶调控区的结合，进而阻碍其对芳香化酶的调控。最终明确SF-1-芳香化酶-甲基化-子宫内膜异位症之间的关系。

子宫内膜异位症是育龄期妇女常见病，病因目前尚不清楚，但被公认是一种雌激素依赖性疾病。芳香化酶是雌激素合成通路的关键合成酶，在异位病灶的间质细胞中高表达，但是在在位内膜的间质细胞中基本没有表达，差异达2659倍。基因表达调控在内异症的发病机制中发挥了重要作用。. 因此，我们对芳香化酶在异位病灶中可能接受的表达调控机制进行了深入的探究。内异症患者腹腔液中高表达的前列腺素 E2（PGE2）激活 MAPK 信号通路，进而增强 P38、JNK 通路下游 ATF2 和 c-Jun 蛋白与芳香化酶的 DNA 启动子区的结合,上调芳香化酶表达；而且在内异症裸鼠模型中，p38、JNK抑制剂能够拮抗PGE2所诱导的芳香化酶的表达上调和异位病灶的增长。其次，我们发现，二甲双胍可以通过激活AMPK信号通路和阻断CRTC2-CREB 转录复合体的形成，抑制PGE2对于StAR和芳香化酶表达的上调作用，进而抑制雌激素的合成，对于内异症具有一定的治疗作用，已经分别发表在J Clin Endocrinol Metab, 2014，99(8)；2015,100(11),IF=5.6和Reprod Sci, 2015, 22(9),IF=2.5。. 此外，我们发现，在异位内膜间质细胞中，高表达的胰岛素样生长因子1（IGF-1）通过激活IGF-1R/PI3K/AKT信号通路，从而促进信号通路下游的转录因子c-Jun和CREB蛋白发生磷酸化，进而增强c-Jun和CREB蛋白与芳香化酶启动子区的结合，在转录水平上调芳香化酶的表达。动物实验证实IGF-1R抑制剂可以缩小IGF-1所诱导的芳香化酶表达的增加，和裸鼠异位症病灶的增长，为内异症的治疗提供了新的治疗靶点，已经发表在J Mol Med, 2016，94(8),IF=4.9。