生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (6): 12-.doi: 10.3969/j.issn.2095-1736.2020.06.012

• 研究报告 • 上一篇    下一篇

miR-29对GnRH基因启动子区特定位点甲基化的影响研究

  

  1. 东华大学 生物科学与技术研究所, 上海 201620
  • 出版日期:2020-12-18 发布日期:2020-12-21
  • 通讯作者: 周宇荀，博士，副教授，研究方向为分子遗传学， E-mail：zhouyuxun@dhu.edu.cn
  • 作者简介:王欣，硕士研究生，研究方向为分子遗传学，E-mail：wangxin1994w@163.com
  • 基金资助:
    国家自然科学基金(31772550)；上海市科委课题（17140903102）

Impact on methylation of the promoter specific sitein GnRH gene with miR-29

  1. Institute of Biological Science and Biotechnology, Donghua University, Shanghai 201620, China
  • Online:2020-12-18 Published:2020-12-21

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摘要： 检测GT1-7细胞中miR-29对GnRH启动子区域甲基化的影响，探究GnRH特定位点的甲基化与其表达量的相关性。采用重亚硫酸盐测序(BSP)结合二代测序技术（NGS）的方法检测GT1-7及miR-29敲除的GT1-7（miR-29KO-GT1-7）细胞中GnRH启动子区7个CpG位点的甲基化程度。根据测序结果，利用CRISPR-dCas9技术在差异显著的CpG6位点特异性富集Dnmt3a、Tet1，并通过二代测序检测该位点甲基化变化，同时利用实时荧光定量PCR技术检测GnRH的表达量，验证二者之间的相关性。结果显示：在miR-29KO-GT1-7细胞中，与对照组相比，在CpG6位点富集Dnmt3a后其甲基化水平上升17%，GnRH的表达水平下降40%（P＜0.01）；在GT1-7细胞中，CpG6位点富集Tet1，可使其甲基化水平下降20%，GnRH的表达水平上升50%（P＜0.05）。结果表明miR-29可通过影响GnRH启动子DNA甲基化水平尤其是特定CpG位点的甲基化程度调控GnRH的表达，为进一步研究解析性发育的调控提供了新的表观遗传机制。

关键词: miR-29, GnRH基因, DNA甲基化, 二代测序

Abstract: The study was to investigate the impact of miR-29 on the methylation state of GnRH promoter region in GT1-7 cells, and to observe the correlation between the specific CpG site of GnRH and its expression. Methylation ration of 7 CpG sites in GnRH promoter in GT1-7 and miR-29 knockout GT1-7 (miR-29KO-GT1-7) cells was determined by bisulfite genomic sequencing (BSP) and the next-generation sequencing. Dnmt3a and Tet1 were recruited at CpG6 site by CRISPR-dCas9, the change of methylation ration was tested by NGS, and the expression of GnRH was detected by real-time PCR. The results showed that overexpression of Dnmt3a at CpG6 site in miR-29KO-GT1-7 cells could make the methylation ration increase by 17% and the expression of GnRH decreased by 40%(P＜0.01); on the other hand, the Tet1 overexpression at this site in GT1-7 cells reduced the methylation level by 20%, while it increased the expression of GnRH by 50%(P＜0.05). Therefore, this study implied that miR-29 could regulate the GnRH expression by modifying the methylation state of the specific CpG site, which provides a novel epigenetic mechanism for the illustration of puberty onset regulation.

Key words: miR-29, GnRH gene, DNA methylation, the next-generation sequencing

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