DNMT3A在子痫前期发生中的深入研究：基因body区甲基化异常的作用和机制

Preeclampsia is a multiple system disorder during human pregnancy, which has become the major causes of maternal and fetal morbidity and mortality. The invasion deficiency of trophoblast cells has been thought as one of the major reasons. It has been demonstrated that genomic DNA methylation is upregulated in preeclamptic placenta, which influences the migration and invasion of trophoblast cells. However, the underlying mechanisms still remain elusive up to now. In our previous work, we reported that DNMT3A downregulation is relevant to preeclampsia. Then, our recent results show that knockdown of DNMT3A obviously increases genomic DNA methylation in trophoblast cells, and FAM46C gene body methylation is negatively regulated by DNMT3A. Moreover, we detected high expression of FAM46C in the clinical samples of preeclamptic placenta. Importantly, FAM46C downregulation can rescue the defects caused by DNMT3A knockdown. Collectively, the findings of our recent study imply that DNMT3A-mediated aberrant FAM46C gene body methylation play important roles in preeclamspsia. Thus, by using trophoblast cell lines in vitro and mouse model in vivo, this project will elucidate the functions and mechanisms of DNMT3A-mediated negative regulation of FAM46C gene body methylation, identify downstream signal pathways regulated by FAM46C in trophoblast cells, clarify the function of DNMT3A and FAM46C in placentation and pregnancy outcome, reveal the correlation between DNMT3A-mediated aberrant FAM46C gene body methylation and preeclampsia. And this study will not only propose a new model for DNMT3A-mediated negative regulation of DNA methylation, but also provide theoretical basis for clinical prevention and treatment for preeclampsia.

滋养细胞侵入不足是子痫前期(PE)发生的主要原因之一，而PE胎盘中DNA甲基化异常升高是导致这一问题的关键因素，但其作用机制尚不明确。我们之前报道了DNA甲基转移酶DNMT3A(3A)在胎盘中的异常低表达对PE发生具有重要作用。有趣的是，我们进一步研究发现与传统认识不同，敲降3A导致了滋养细胞基因组DNA甲基化升高，FAM46C(46C)基因body甲基化程度明显增加且其蛋白在PE胎盘中高表达。重要的是，敲降46C能完全恢复3A缺失对滋养细胞迁移侵袭的影响。这些结果提示3A介导的46C甲基化异常调控在PE中扮演了关键角色。在此基础上，本课题将利用滋养细胞、小鼠囊胚等模型深入探讨3A负调控46C基因body甲基化的功能和机制，鉴定46C作用于滋养细胞的关键下游通路，揭示3A介导的46C甲基化异常调控在PE中的重要作用。本项目有望提出3A负调控DNA甲基化的新模式，为PE临床诊治提供新的思路。

滋养细胞侵入不足是早发型子痫前期(EO-PE)发生的主要原因之一，而EO-PE胎盘中DNA甲基化异常升高是导致这一问题的关键因素，但其机制尚不明确。我们之前报道了DNA甲基转移酶DNMT3A在胎盘中异常低表达对EO-PE发生具有重要作用，但其对滋养细胞基因组DNA甲基化的调控功能并不清楚。本项目以DNMT3A对滋养细胞基因组DNA甲基化的调控作用为切入点，深入研究其中的生物学机制，进而揭示DNMT3A异常表达在早发型子痫前期胎盘滋养细胞基因组DNA甲基化异常升高中的作用机制。首先，我们发现与正常孕晚期胎盘相比，早发型子痫前期胎盘基因组DNA甲基化水平显著升高；敲降DNMT3A导致滋养细胞基因组DNA甲基化水平显著升高；甲基化芯片结果显示，敲降DNMT3A导致甲基化升高的位点主要位于gene body区(61/121)。FAM46C是受DNMT3A调控的差异甲基化基因，敲降DNMT3A后，FAM46C gene body甲基化水平升高，同时转录和蛋白水平升高。功能回补实验显示，敲降FAM46C能够完全恢复抑制DNMT3A对滋养细胞迁移和侵袭功能的影响。随后的机制研究证明，敲降DNMT3A虽然不会改变DNMT1和DNMT3B在滋养细胞中的蛋白水平，但会增加其在FAM46C gene body区的富集，这一结果提示我们DNMT3A减少导致DNMT1和DNMT3B在靶基因的定位增加可能是导致滋养细胞基因组DNA甲基化改变的原因。随后RNA-seq分析敲降DNMT3A和过表达FAM46C对滋养细胞转录水平的影响，结果发现差异表达基因显著富集于细胞外基质受体相互作用和黏着斑通路，体外实验显示，敲降DNMT3A和过表达FAM46C能够显著抑制滋养细胞对Ⅳ胶原的粘附作用。最后我们对临床样本进行检测发现，与正常孕晚期和早产胎盘相比，早发型子痫前期胎盘绒毛外滋养细胞的FAM46C表达显著增加，粘附相关基因整合素β4的表达显著降低。因此，本项研究揭示了DNMT3A在早发型子痫前期胎盘基因组DNA甲基化异常中的重要作用，扩展了表观遗传调控在疾病发生中的作用机制，并为早发型子痫前期的靶向治疗提供了新思路。