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叶酸缺乏抑制
Brachyury 表达的表观调控机制及其对拟胚体分化的影响Epigenetic regulation mechanism of folic acid deficiency inhibiting
Brachyury expression and its effect on differentiation of embryoid bodies- 陆军军医大学学报 2022年44卷第14期 页码：1401-1409
- 作者机构：
1.100020 北京，首都儿科研究所，儿童发育营养组学北京市重点实验室
2.100026 北京，朝阳区妇幼保健院儿科
3.266033 山东 青岛，青岛市海慈医疗集团泌尿外科
- 作者简介：
[常韶燕，E-mail：changsyan_2001@163.comCHANG Shaoyan, E-mail: changsyan_2001@163.com
- 基金信息：国家自然科学基金青年科学基金(82001555)
DOI：10.16016/j.2097-0927.202201119
中图分类号： R321.4;R345.638;R394.1收稿：2022-01-14，
修回：2022-03-16，
纸质出版：2022-07-30
- 稿件说明：
移动端阅览
常韶燕, 郭九叶, 陈燕, 等. 叶酸缺乏抑制
Brachyury 表达的表观调控机制及其对拟胚体分化的影响[J]. 陆军军医大学学报, 2022,44(14):1401-1409.Shaoyan CHANG, Jiuye GUO, Yan CHEN, et al. Epigenetic regulation mechanism of folic acid deficiency inhibiting
Brachyury expression and its effect on differentiation of embryoid bodies[J]. Journal of Army Medical University, 2022, 44(14): 1401-1409.常韶燕, 郭九叶, 陈燕, 等. 叶酸缺乏抑制
Brachyury 表达的表观调控机制及其对拟胚体分化的影响[J]. 陆军军医大学学报, 2022,44(14):1401-1409. DOI： 10.16016/j.2097-0927.202201119.Shaoyan CHANG, Jiuye GUO, Yan CHEN, et al. Epigenetic regulation mechanism of folic acid deficiency inhibiting
Brachyury expression and its effect on differentiation of embryoid bodies[J]. Journal of Army Medical University, 2022, 44(14): 1401-1409. DOI： 10.16016/j.2097-0927.202201119.
摘要
目的
2
探讨叶酸缺乏抑制发育关键基因
Brachyury
表达的表观调控机制及对拟胚体(embryoid bodies，EBs)分化的影响。
方法
2
建立叶酸缺乏的小鼠胚胎干细胞(embryonic stem cell，ESCs)分化为EBs的细胞模型。根据叶酸浓度分为3组：正常组，叶酸缺乏组和叶酸补充组。采用massARRAY和RT-PCR分别检测EBs中
Brachyury
基因的甲基化和表达水平，明确叶酸缺乏对
Brachyury
基因表达的影响；RT-PCR和ChIP-qPCR分别检测DNA甲基化酶的表达和组蛋白甲基化修饰，探讨叶酸缺乏对
Brachyury
基因的表观调控机制；检测多潜能markers和三胚层markers的表达，探讨发育关键基因
Brachyury
表达异常对EBs分化的影响。
结果
2
① 在叶酸缺乏的ESCs分化为EBs模型中
Brachyury
表达显著降低，叶酸缺乏组的相对表达量为正常组的(0.19±0.08)倍，叶酸补充后上升至正常组的(0.62±0.14)倍。EBs中
Brachyury
基因启动子区甲基化
水平从正常组的(30.75±0.50)%升高至叶酸缺乏组的(37.93±1.24)%，补充叶酸后则降低至(31.88±2.67)%。②DNA从头甲基化酶
DNMT
3
b
表达升高，叶酸缺乏组其相对表达量为正常组的(1.59±0.31)倍。叶酸缺乏的EBs中H3K27me3修饰增加，正常组、叶酸缺乏组、叶酸补充组的相对富集程度分别为(0.22±0.03)、(0.32±0.02)、(0.24±0.01)。③在叶酸缺乏ESCs分化为EBs后，细胞形态发生改变：EBs小球体积变小，形态不规则；叶酸补充后EBs小球体积接近正常，呈规则的球体结构。④ESCs多潜能性markers包括
Nanog
和
Sox
2表达显著上调，其相对表达量在叶酸缺乏组的EBs中分别为正常组的(3.18±0.95)和(4.23±1.59)倍；叶酸缺乏的EBs中外胚层marker
Fgf
5和中胚层marker
Gsc
表达量显著下调，分别为正常组的(0.42±0.04)倍和(0.61±0.20)倍。
结论
2
叶酸缺乏通过DNA甲基化和组蛋白H3K27甲基化的双重表观调控作用，引起
Brachyury
基因的表达显著下调，最终导致拟胚体EBs的分化受阻。
Abstract
Objective
2
To investigate the epigenetic regulation mechanism of folic acid deficiency in inhibition of the expression of
Brachyury
a key developmental gene
and its effect on the differentiation of embryoid bodies (EBs).
Methods
2
Firstly
the cell model of folic acid-deficient embryonic stem cells (ESCs) differentiated into EBs was established. According to the folate level
they were divided into normal group
folate deficiency group and folate supplement group. MassARRAY system and RT-PCR were used to detect the methylation and expression level of
Brachyury
gene in EBs to clarify the effect of the deficiency on the expression. RT-PCR and chromatin immunoprecipitation (ChIP)-qPCR were used to detect the expression of DNA methylase and histone methylation modification
respectively to explore the epigenetic regulation mechanism of folic acid deficiency on
Brachyury
expression. At the same time
the expression levels of pluripotent markers and triploblastic markers were detected to explore the effect of abnormal expression of
Brachyury
on EBs differentiation.
Results
2
① In the differentiation of ESCs into EBs under folic acid deficie
ncy
the expression level of
Brachyury
was decreased significantly
only 0.19±0.08 times of that in the normal group
and folate supplementation increased the level to 0.62±0.14 times of that of the normal group. The methylation level of promoter region of
Brachyury
in EBs was increased from (30.75±0.5)% in the normal group to (37.93±1.24)% in the folic acid-deficient group
and decreased to (31.88±2.67)% after folic acid supplementation. ② The expression level of
DNMT
3
b
DNA de novo methylase
in folic acid-deficiency group was increased by 1.59±0.31 times higher than that of normal group. H3K27me3 modification was enhanced in the EBs with folate deficiency
and the relative enrichment level was 0.22±0.03 in the normal group
0.32±0.02 in the folate deficiency group
and 0.24±0.01 in the folate supplement group. ③After the differentiation of folic acid-deficient ESCs into EBs
the cell morphology was significantly changed with the smaller EBs spheres and irregular shape. Folic acid supplementation resulted in normal volume and regular shape of EBs spheres. ④ ESCs pluripotent markers
Nanog
and
Sox
2
were significantly up-regulated
and their relative levels in EBs of folate deficiency group was 3.18±0.95 and 4.23±1.59 times higher than those of normal group. The expression of ectodermal marker
Fgf
5 and mesodermal marker
Gsc
in folic acid-deficient EBs were significantly down-regulated
which was 0.42±0.04 times and 0.61±0.20 times in folic acid-deficient EBs when compared with those in normal EBs
respectively.
Conclusion
2
Through the dual epigenetic regulation of DNA methylation and histone H3K27 methylation
folic acid deficiency causes significant down-regulation of
Brachyury
and ultimately leads to the blocked differentiation of EBs.
关键词
Keywords
references
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