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