miR-29对GnRH基因启动子区特定位点甲基化的影响研究

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

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。

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.