子宫内膜异位症患者卵巢颗粒细胞EZH2通过组蛋白甲基化修饰导致女性不孕的作用机制研究

Histone methylation is a hot topic at present, but few work was reported on endometriosis. We recently found that the expression of histone methyltransferase EZH2 in granulosa cells of endometriotic patients was significantly lower than that in normal women, and interfering with EZH2 in granulosa cells could increase progesterone synthesis, induce cell senescence and apoptosis. EZH2 combines with the genomic DNA of target genes, and modifies adjacent histone methylation, resulting in inhibition of gene transcription. Our previous results of CHIP-Seq and CHIP-PCR confirmed that EZH2 could bind to genomic DNA of CYP11A1, a key catalytic enzyme of sex homone, and P16 and P21, biomarkers of cell senescence. EZH2 Interference could increase CYP11A1, P16 and P21. These data indicated that CYP11A1, P16 and P21 are target genes of EZH2. In this research, we propose that the downregulated EZH2 of granulosa cells in patients with endometriosis leads to the decrease of methylation of genomic DNA of CYP11A1, P16 and P21, and affects sex hormone synthesis and cell senescence and apoptosis, which cause infertility of endometriotic patients. We would like to overexpress or interfere EZH2 in human primary cultured granulosa cells, detect progesterone level, cell senescence and apoptosis. We plan to construct endometriotic mice model and transgenic mice with EZH2 conditional knockout in granulosa cell, and study the effect of EZH2 on fertility of mouse. This project will identify the mechanism of EZH2 function in infertility of patients with endometriosis, and provide potential targets for diagnosis and treatment of the disease.

组蛋白甲基化修饰是目前的研究热点，但在内异症的报道甚少。我们发现组蛋白甲基化转移酶EZH2在内异症患者颗粒细胞的表达显著低于正常女性,干扰颗粒细胞EZH2可促进孕酮合成、诱导细胞衰老和凋亡。EZH2与靶基因DNA结合，甲基化修饰临近组蛋白从而抑制基因转录。我们CHIP-Seq及CHIP-PCR证实EZH2可与雌孕激素合成关键催化酶CYP11A1、衰老蛋白P16与P21基因组DNA结合，且干扰EZH2可上调CYP11A1、P16和P21。我们提出内异症患者颗粒细胞EZH2下调，导致CYP11A1、P16和P21基因组蛋白甲基化修饰减弱，影响激素合成和细胞衰老凋亡，导致患者生育力下降。我们拟过表达/干扰EZH2，检测孕酮水平、细胞衰老凋亡变化；构建内异症小鼠和颗粒细胞条件性敲除EZH2小鼠，研究EZH2对小鼠生育力的影响。本项目将明确EZH2在内异相关性不孕的作用机制，为疾病诊治提供潜在靶点。

子宫内膜异位症是一种常见的妇科疾病，以慢性盆腔疼痛和不孕为特征，其导致不孕的机制仍不清楚。黄素化未破裂卵泡综合征是最常见的排卵功能障碍类型，是子宫内膜异位症相关不孕的原因，包括回收和成熟卵母细胞数量减少。排卵由卵泡微环境中产生的黄体生成素和旁分泌信号控制。一般来说，子宫内膜异位症卵泡液中白细胞介素IL-1β升高，从而通过激活ERK1/2和CCAAT/EBP-β途径增加排卵信号。然而，在子宫内膜异位症患者中，IL-1β并不会促进排卵。为了阐明子宫内膜异位症排卵功能障碍的机制，我们分析了氧化应激和IL-1β表达对子宫内膜异位卵泡的影响。我们发现氧化应激降低了子宫内膜异位症卵巢颗粒细胞（GC）中EZH2的表达和H3K27Me3的水平。小鼠卵巢GCs中选择性Ezh2耗竭通过干扰卵丘-卵母细胞复合体的扩张和减少表皮生长因子样因子的表达来降低生育力。GCs的基因表达和H3K27Me3 ChIP测序（ChIP Seq）揭示了IL-1受体2（IL-1R2），这是一种高浓度的IL-1β受体，在排卵期间抑制IL-1β介导的炎症级联反应，是EZH2-H3K27Me3轴的关键靶基因。此外，加入IL-1β并不能在Ezh2敲除后恢复排卵，这表明IL-1R2在子宫内膜异位症中具有重要作用。因此，我们的发现表明，降低GC中的EZH2和H3K27Me3通过增加IL-1R2表达来抑制排卵信号，这可能最终导致子宫内膜异位症相关的不孕。.卵泡生成、排卵和黄体化是连续而复杂的过程。GCs中EZH2组蛋白甲基转移酶活性的不及时降低刺激IL-1R2表达，并通过阻断IL-1β信号传导干扰排卵级联，最终导致子宫内膜异位症相关不孕。尽管LH激增在诱导排卵中起着重要作用，但我们证实组蛋白甲基化修饰激活的IL-1R2上调也会干扰排卵过程。总之，我们的研究对卵泡微环境进行了全面的观察，以阐明组蛋白甲基化降低导致排卵功能障碍和子宫内膜异位症相关不孕的新机制。具体而言，组蛋白甲基化降低上调了高亲和力抑制受体IL-1R2，并抑制了IL-1β信号传导，从而破坏排卵级联，最终减少了排卵相关基因的表达。我们建议，在治疗子宫内膜异位症的临床实践中，服用非甾体抗炎药，特别是PGE2合成抑制剂可能是合理的。