子痫前期滋养细胞中TFPI-2的DNA甲基化与组蛋白修饰的特点及调控机制

Our previous studies showed that alternation of TFPI-2 expression in preeclampsia may be associated with hypoxia in local trophoblast of placenta. However, the reason and regulated mechanism of the change of TFPI-2 expression in preeclamspsia remain to be elucidated. We hypothesize that DNA methyltransferases,histone acetylation and histone methylation may regulate DNA methylation, which lead to hypomethylation of TFPI-2 gene. As a result, TFPI-2 expression increase and effect the function of trophoblasts such as migration, invasion, and proliferation/apoptosis, which in turn cause the pathogenesis of preeclampsia. In order to validate the hypothesis we design this proposal. We plan to carry out the study from three aspects including case control, in vitro cells experiment,intervention of transfection technique and 5aC. First, real time PCR, Western Blot,MassARRAY and chromatin immunoprecipitation (ChIP) assay will be used to detect the expression of TFPI-2 mRNA and protein, DNA methyltransferases mRNA and protein, DNA methylation, histone acetylation and histone methylation in placenta of women with preeclampsia and normal pregnancy. Second, JEG-3 and BeWo trophoblastic cell line will be cultured with normoxia and anoxia condition. Level of above index will be measured to study the effects of hypoxia on the expression of DNA methylation, histone acetylation, histone methylation and TFPI-2. Finally, transfection technique and 5-Azacytidine(5aC) will be used to up-regulate or down-regulate the activity of DNMTs in trophoblast, then alternations of above index and effects of the trophoblast's function will be examined.Therefore, we attempted to explore the possible pathogenesis of preeclampsia and provide a new thread of prevention and treatment.

本课题组前期工作发现子痫前期TFPI-2的表达改变可能与胎盘局部滋养细胞缺氧有关，但具体机制不明。我们提出假说：DNA甲基化和组蛋白修饰（甲基化和乙酰化）可能调控TFPI-2的表达，从而影响滋养细胞的功能，与子痫前期的发病有关。本课题拟从病例对照，体外细胞实验，转染/5aC干预三个层面开展研究：首先，采用RT-PCR、Western Blot、MassARRAY、ChIP检测胎盘TFPI-2、DNMTs、DNA甲基化、组蛋白甲基化和乙酰化的表达；其次，常氧和缺氧培养JEG-3、BeWo滋养细胞株，研究缺氧时滋养细胞DNA甲基化、组蛋白修饰（甲基化和乙酰化）的表达改变及其对TFPI-2表达的影响。最后，采用转染/5aC干预滋养细胞中DNMTs活性，进一步研究DNMTs参与的TFPI-2 DNA甲基化对滋养细胞功能（侵袭、迁移、增殖、凋亡）的影响，探讨其在子痫前期发病中的作用。

本课题从病例对照，体外细胞实验，转染/5aC干预三个层面研究了TPFI-2的DNA甲基化在子痫前期发病中的作用。研究发现：子痫前期组胎盘组织中TFPI-2基因的表达水平明显升高，DNMT1的表达水平显著下降，且与病情呈正相关，证实胎盘滋养细胞的局部缺氧导致了TFPI-2的DNA甲基化状态发生改变。在JEG-3滋养细胞运用si-RNA特异性敲除DNMT-1基因后，TFPI-2表达水平明显的升高；特异性质粒转染上调DNM-1基因后，但相应的TFPI-2表达未见明显下降，证实DNMT1调控着DNA甲基化的表达水平，但不是唯一的调控机制。本研究结果丰富了子痫前期的发病学说，为进一步研究子痫前期的发病机制提供了基础。