ASPP2调控妊娠期高血压疾病胎盘滋养细胞自噬水平升高的表观遗传学机制研究

Placental dysfunction which induced by the elevated levels of trophoblast autophagy is an important key link of Hypertension disorder complicating pregnancy(HDCP), our preliminary studies indicated that DNA methylation is an significant mechanism of HDCP, but what puzzled us is that why diverse levels of DNA methylation coexist in different genes under the same condition. There existed a combined effect between Histone methylation and DNA methylation, also p53 apoptosis stimulating protein 2(ASPP2) is rich in CpG islands within its promoter region and regulated by transcription factors. Therefore, we speculate that histone methylation and DNA methylation could interact with each other to recruit transcription factors to regulate the expression of ASPP2 is an important mechanism in HDCP induced by trophoblast autophagy. To vertify this hypothesis, first of all, Western blot detect the expression levels of ASPP2 in placental tissues; Detected the autophagy related gene expression after silience and over-express ASPP2 gene, which to clarify that the effect of ASPP2 in trophoblast autophagy; Morever, block-oriented strategy is used from the perspective of the bi-directional regulation of histone and DNA methylation to explore their effect on the target gene after their bacance model is broken; Last but not least, screen and confirm Sp1 and E2F1 are the important transcription factors of HDCP, then transfect their activated and deactivated mutant accompany by DNMT1 vector, so as to verify the interaction between the two bargeted genes, and reveal the mechanism of histone methylation and DNA methylation which recruit transcription factors to regulate ASPP2 to lead to trophoblast autophagy. In this way, we can provide theoretical basis for the prevention and treatment of HDCP.

细胞自噬水平升高引起胎盘功能障碍是妊娠期高血压疾病(HDCP)的中心环节，前期研究表明DNA甲基化是其重要机制，但为何同条件下不同基因DNA甲基化高低并存仍有争议。组蛋白和DNA甲基化具有协同作用，且p53凋亡刺激蛋白2(ASPP2)启动子区富含CpG岛并受转录因子调控，故推测：组蛋白和DNA甲基化相互作用招募转录因子调控ASPP2是滋养细胞自噬的重要机制。为了验证该假说，免疫印迹等检测胎盘组织中ASPP2的表达，并使其沉默和过表达后检测自噬相关基因的变化，探讨ASPP2在细胞自噬的作用；采用阻断为主的策略从组蛋白和DNA甲基化双向调控入手，探讨各自平衡模式被打破后对靶基因的影响；筛选并确定Sp1和E2F1是HDCP的重要转录因子，并将其持续失活和激活突变体分别与甲基化调控酶等质粒和/或共转染细胞，揭示组蛋白和DNA甲基化招募转录因子调控ASPP2的机制，为防治HDCP提供依据。

背景：滋养细胞自噬水平升高引起胎盘功能障碍是妊娠期高血压疾病(HDCP)的中心环节，前期研究表明DNA甲基化是其重要机制，组蛋白和DNA甲基化具有协同作用，且p53凋亡刺激蛋白2(ASPP2)启动子区富含CpG岛并受转录因子调控，但其在HDCP滋养细胞自噬水平升高中的分子机制尚不清楚。.内容：收集HDCP和同期正常分娩孕妇(PC)胎盘组织，qRT-PCR和Western blot检测ASPP2的表达，并使其沉默和过表达后检测自噬相关蛋白的变化，探讨ASPP2在细胞自噬中的作用；采用阻断为主的策略从组蛋白和DNA甲基化双向调控入手，探讨各自平衡模式被打破后对靶基因的影响；筛选并确定Sp1和E2F1是HDCP的重要转录因子，并将其持续失活和激活突变体分别与甲基化调控酶等质粒和/或共转染细胞，揭示组蛋白和DNA甲基化招募转录因子调控ASPP2的机制。.结果：与对照组比较，HDCP胎盘和缺氧诱导的HTR8/SVneo细胞中ASPP2表达升高，同时自噬增强。此外，ASPP2过表达促进滋养细胞自噬，干扰呈现相反作用。表观遗传修饰分析表明，抑制DNA甲基转移酶1（DNMT1）和组蛋白甲基转移酶（G9a）的表达共同介导了ASPP2启动子区DNA低甲基化和低H3K9me2水平，导致ASPP2上调和细胞自噬的激活。进一步研究表明，E2F1与 ASPP2启动子的结合可以增强该基因在HDCP中的转录，这得益于DNMT1和G9a 之间的相互作用。 .该项目共发表SCI 2篇，中文核心7篇。.意义：该课题的实施有利于阐明HDCP的发病机制，寻找致病环节，确定关键靶点，为HDCP靶向治疗提供新的干预途径，为HDCP这一全球重大疾病的防治工作提供更多研究资料。