中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (2): 361-368.doi: 10.4103/1673-5374.244799

• 原著:脊髓损伤修复保护与再生 • 上一篇    

MGMT基因在显性脊柱裂大鼠中的表达

  

  • 出版日期:2019-02-15 发布日期:2019-02-15

MGMT is down-regulated independently of promoter DNA methylation in rats with all-trans retinoic acid-induced spina bifida aperta

He-Nan Zhang 1 , Yi Guo 1 , Wei Ma 1 , Jia Xue 1 , Wei-Lin Wang 2 , Zheng-Wei Yuan 1   

  1. 1 Key Laboratory of Health Ministry for Congenital Malformation, Shengjing Hospital, China Medical University, Shenyang, Liaoning Province, China
    2 Department of Pediatric Surgery, Shengjing Hospital, China Medical University, Shenyang, Liaoning Province, China
  • Online:2019-02-15 Published:2019-02-15
  • Contact: Zheng-Wei Yuan, PhD, yuanzw@hotmail.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81671469, 81171072 (to ZWY); the National Basic Research Program of China (973 Program), No. 2013CB945402 (to ZWY); the Program for Liaoning Innovative Research Team in University of China, No. LT2013016 (to ZWY).

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摘要：

O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)作为一种DNA修复酶介导了最常见的DNA修复作用，对于该基因的研究在其他先天性畸形中已见报道，但其在神经管畸形的作用中报道较少。实验以此探讨MGMT在胚胎早期以及不同胚胎时期mRNA的表达情况和启动子的甲基化水平以及与神经管畸形的关系。(1)采用全反式维甲酸于胚胎第10天(E10)灌胃处理建立显性脊柱裂模型大鼠，正常对照组给予同等剂量的橄榄油灌胃处理，应用Western-blot在显性脊柱裂模型组中检测γ-H2A.X蛋白表达评估DNA损伤情况，并应用Real-time PCR方法检测各组MGMT mRNA表达；(2)正常对照组MGMT mRNA表达随着胚胎日数的增加而下降，并且在E11至E14时显著降低，在E18时达到最小；(3)在显性脊柱裂模型组中，γ-H2A.X 蛋白显著增加，并且MGMT mRNA表达在E14，E16和E18显著降低；(4)通过亚硫酸氢盐测序PCR(BSP)分析MGMT启动子甲基化水平结果显示，MGMT启动子中的几乎所有CpG位点在显性脊柱裂模型组和对照中均未被甲基化，并且在E14或E18胚胎中两组之间没有检测到甲基化水平的显著差异；(5)上述结果数据表明，全反式维甲酸诱导的显性脊柱裂模型胎鼠中存在DNA损伤，MGMT mRNA表达下调，并且这种下调不依赖于启动子DNA甲基化。

orcid: 0000-0003-0967-9462 (Zheng-Wei Yuan)

关键词: 神经管缺损, 脊柱裂, 脊髓, 全反式维甲酸, O6-甲基鸟嘌呤DNA甲基转移酶(MGMT), 基因表达, dna甲基化, 启动子, 亚硫酸氢盐序列PCR, 神经再生

Abstract:

O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day (E) 10, whereas normal control rats re¬ceived the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methyla¬tion demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is indepen¬dent of promoter DNA methylation.

Key words: nerve regeneration, neural tube defects, spina bifida aperta, spinal cord, all-trans retinoic acid, O6-methylguanine DNA methyl¬transferase, gene expression, DNA methylation, promoter, bisulfite sequencing polymerase chain reaction, neural regeneration