叶酸缺乏对Gcm1影响Wnt/β-catenin信号通路的调控机制及在神经管畸形中的作用研究

Ectopic expression of glial cell loss factor Gcm1 produces NTD, however, the mechanism is unclear. Wnt signaling pathway is the key pathway to determine the closure of neural tube. It is not known whether the regulation of Gcm1 affect Wnt/β-catenin pathway by the early folic acid deficiency in the embryo. In samples from NTD with low folate level, aberrant Gcm1 levels were observed along with enhanced binding with β-catenin in our previous studies. Based on the cell model with folate deficiency, we would like to investigate the relationship between Gcm1 expression level and Wnt/β-catenin pathway, making further study on the molecular mechanism of Gcm1 affecting the pathway with the technique of CHIP-qPCR、Co-IP、FOP&TOP flash. The epigenetic modification of Gcm1 activating will be confirmed by QE-MS mass spectrometry and methylation sequencing. Furthermore, the validation of above mechanism will be made in NTD mouse model of folate metabolism and human NTD samples combined with Nanostring technology. Therefore, it will attempt us to clarify NTD etiology from the perspective of "activation of Gcm1 by folic acid deficiency -Wnt / β-catenin" and advance the scientific understanding of early folic acid on epigenetic programming, promoting reproductive health.

异位表达胶质细胞缺失因子Gcm1产生NTD，作用机制不清。Wnt信号通路是决定神经管闭合的关键通路。胚胎早期叶酸缺乏是否介导Gcm1调控Wnt信号通路未见报道。我们已证实低叶酸NTD标本中Gcm1特异性增高，并与Wnt信号通路中β-catenin交互作用增强。现拟通过叶酸缺乏细胞模型明确Gcm1表达水平对Wnt/β-catenin信号通路活性调控影响；采用CHIP-qPCR、Co-IP、FOP&TOP flash等探讨Gcm1影响Wnt信号通路的具体分子机制；利用QE-MS质谱、甲基化测序等确认叶酸缺乏介导Gcm1高表达的表观修饰方式；最后结合Nanostring技术在叶酸代谢障碍NTD小鼠模型及低叶酸人标本中验证上述机制，因而阐明在NTD病因中是否存在“叶酸缺乏通过表观修饰激活Gcm1-Wnt/β-catenin”分子病理路径，推进对早期叶酸影响表观发育编程作用的科学认识，促进生殖健康。

背景: 基于叶酸缺乏C57BL/6小鼠胚胎干细胞表达谱芯片探讨叶酸缺乏下神经管畸形（Neural tube defects, NTDs）易感基因的转录差异变化；研究Gcm1如何在叶酸缺乏下调控Wnt/β-catenin信号通路而参与NTDs的发生；最后从表观遗传学修饰角度解释叶酸缺乏导致C57BL/6小鼠胚胎干细胞中Gcm1表达显著升高的具体分子调控机制，从而为叶酸缺乏引起NTDs提供新的理论依据。.研究内容: 构建叶酸缺乏细胞以及正常对照胚胎干细胞模型，行表达谱测序，将287个NTDs易感基因与差异表达基因取交集，选出差异最显著的易感基因Gcm1；采用Co-IP在细胞水平检测Gcm1蛋白与ß-catenin蛋白在叶酸缺乏下相互结合；沉默Gcm1基因，检测ß-catenin，Tcf4与下游靶蛋白Axin2表达的变化；过表达Gcm1基因，分析Gcm1蛋白与ß-catenin、Tcf4之间的交互作用及其对Wnt/β-catenin信号通路的调控作用；检测CBP乙酰化转移酶与Gcm1蛋白的结合情况；通过ChIP-qPCR验证Gcm1基因启动子区组蛋白乙酰化表达改变；选择低叶酸NTDs标本及配对正常流产胚胎标本，采用 Western Blot技术检测脑组织以及心脏、肺和肌肉组织中GCM1蛋白表达水平；分析叶酸缺乏影响GCM1蛋白调控经典Wnt/β-catenin信号通路与NTDs的产生关联性。.结果: 1.表达谱芯片数据显示NTDs易感基因Gcm1升高16倍，Co-IP结果显示叶酸缺乏六代培养的C57BL/6 mESC中Gcm1蛋白和ß-catenin蛋白的结合增加（P < 0.05）。转染siRNA片段沉默Gcm1基因引起Tcf4、Axin2表达下降（P < 0.05）。过表达Gcm1质粒，Co-IP检测到过表达的Gcm1蛋白与ß-catenin蛋白、Tcf4蛋白结合增加（P < 0.05）; 2.证实Gcm1表达升高主要是通过CBP乙酰化转移酶介导的Gcm1基因启动子区H4乙酰化增加所致；人低叶酸NTDs脑组织标本验证发现GCM1蛋白表达均增加且GCM1蛋白与ß-catenin蛋白结合显著增加（P < 0.05）。.结论及意义: 叶酸缺乏主要通过CBP依赖的H4组蛋白乙酰化促进Gcm1/ß-catenin/Tcf4复合物形成激活经典Wnt信号通路参与NTDs发生。