叶酸缺乏介导PEG10基因印记改变经TGF-β通路在神经管畸形发生中的作用及机制研究

PEG10, a maternally imprinted gene, is thought to be involved in the regulation of embryonic development by antagonizing the TGF-βpathway. Our recent study demonstrated that PEG10 methylation levels in differentially methylation region (DMR), a core regulatory region of gene imprinting, were higher than that in the control group in NTDs brains with folate deficiency, accompanied with transcription reduction, however, whether PEG10 imprint modified by folate can affect neural tube closure though disturbing TGF-βpathway remains unclear. Based on the NTDs sample and mouse model with folate deficiency in the present program, we are going to use Massarray, Epiq, SPR, RT-PCR et al technologies to study following questions: the characteristic of imprinting modification of PEG10, the time curve of imprinting establishment and the mechanism underlying imprinting affecting transcription regulation; to analysis the correlation among folate insufficiency, aberrant imprinting modification and disturbance of TGF-βpathway; Mouse embryonic stem cell will be treated with small interfering RNA and folate supplement to test and explore whether PEG10 gene imprinting alternation affected by folate insufficiency disturbs TGF-β pathway in neural tube development. It is respected that the study will explain the molecular mechanism of aberrant folate-related metabolism induced NTDs in the aspect of dysregulation of imprint modification, and further offer the experimental evidence to deepen the understanding the role of PEG10 in normal development programming and health embryonic growth.

母系印记基因PEG10通过拮抗TGF-β通路参与早期胚胎发育调控。课题组前期研究发现PEG10甲基化差异区DNA甲基化水平在人类低叶酸神经管畸形（NTDs）病例显著增高，表达降低，而低叶酸状态下该基因印记改变是否经TGF-β通路引起神经管发育异常尚未见报道。本研究在前期建立NTDs标本库及低叶酸小鼠模型基础上，拟用Massarray、Epiq、SPR及RT-PCR等技术，深入研究胚胎发育过程中PEG10印记建立特点、时间曲线，调控转录的机制及叶酸缺乏对该基因印记修饰及TGF-β通路的影响，分析低叶酸、印记修饰及TGF-β通路改变的关系；并用RNA干扰等处理低叶酸小鼠胚胎干细胞模型进行验证，探寻低叶酸状态该基因印记改变是否通过TGF-β通路引起神经管发育异常，结果将从印记基因调控角度进一步阐明叶酸缺乏引起NTDs的分子机制，为深入理解PEG10基因在早期胚胎神经发育中的作用提供新的实验依据。

叶酸相关的一碳单位代谢为包括DNA和组蛋白的甲基化修饰提供甲基，叶酸相关代谢紊乱引起的基因组印记异常与神经管畸形(Neural tube defects, NTDs)的发生有密切的关联，其中甲基化差异区（DMR）作为基因组印记调控的核心区域对叶酸缺乏异常敏感，但叶酸缺乏引起此区域表观修饰的改变在NTDs发生中的机制尚不完全明确。本项目利用课题组前期建立的NTDs标本库及包括差异甲基化的细胞模型、叶酸缺乏的小鼠胚胎干细胞模型及缺叶酸饲料喂养的小鼠动物模型在内的多个细胞及动物模型，开展体内外实验以研究叶酸缺乏对母本印记基因PEG10印记建立的影响以及在NTDs发生中的作用和机制。研究首先发现低叶酸水平的NTDs脑组织中PEG10基因的转录及表达水平显著低于正常对照组，利用Massarray技术平台对其DMR区进行甲基化检测显示NTDs样本中甲基化水平异常增高，与NTDs的发生显著相关，提示PEG10印记紊乱可能是NTDs发生的危险因素。利用甲基化转移酶抑制剂5-AZA构建的差异甲基化的细胞模型证实PEG10印记簇DMR区的甲基化水平与该印记簇内包括PEG10在内多个印记基因的转录水平呈显著负相关，提示叶酸相关代谢异常引起的DMR区异常印记参与印记簇内基因转录水平的调控。调亡分析显示PEG10表达下调的NTDs脑组织样本中存在过度调亡的发生，应用流式细胞分析术对差异甲基化的细胞模型进行分析，发现处理组PEG10表达的激活与调亡抑制相关联，提示叶酸缺乏可能通过干扰PEG10基因印记建立，抑制其表达进而异常增加细胞调亡而参与NTDs的发生。本项目进一步证实叶酸相关代谢紊乱介导的基因组印记异常参与NTDs的发生，为围孕期叶酸补充预防NTDs提供了新的实验依据。