利用目標甲基化細胞模型來探求MAEL基因特定啟動子序列甲基化後影響的下游調控基因
鄭裕生、黃詩凱、陳幸儀、林永明
國立成功大學醫學院臨床醫學研究所、國立成功大學醫學院附設醫院泌尿部2
Investigating the downstream candidate genes after hypermethylation of the MAEL promoter region in targeted DNA methylation(TDM) cell model
Yu-Sheng Cheng, Shi-Kae Wee, Hsing-Yi Chen, Yung-Ming Lin
Department of Urology, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan
Objective:
MAEL was known to be essential in spermatogenesis because MAEL knockout mice are sterile presented with intra-testicular meiotic arrest. In our previous study, dysregulated methylation of MAEL promoter may lead to de-repression of LINE-1, which may contribute to one of the causes of spermatogenic failure in infertile men. We successfully established the targeted DNA methylation(TDM) in MAEL promoter region(from -188 to +294) in human H358 cell line for investigating the detailed mechanism after MAEL promoter methylation. In this study, we aimed to explore the downstream candidate genes of targeted DNA methylation(TDM) in MAEL promoter by next-generation sequencing method.
Materials and methods:
After validating the targeted MAEL promoter methylation in NCI-H358 cell models, we sent the TDM cells and un-methylated cells for high throughput next-generation sequencing (NGS) analysis. The selecting criteria were set as protein-encoding genes, FPKM>1, and known genes related to spermatogenesis. The significance of the difference between the TDM cells and un-methylated cells was set at P < 0.05.
Results:
We set the criteria for protein-encoding genes and FPKM>1, 120 protein-encoding genes were initially selected. After the intensive literature review, fourteen genes were regarded as downstream target genes after MAEL promoter methylation. CYP1A1 and SPIN2B have been shown to have significantly higher transcript expression in TDM cells versus un-methylated cells. On the other hand, another 12 genes have significantly lower transcript expression in TDM cells versus un-methylated cells, including SMARCA1, MKI67, PRC1, AURKB, SPANXB1, SGK1, CDH2, ASF1B, ESYT3, EGF, BIRC3, and MICALCL.
Conclusions:
We reported 14 candidate genes that might be downstream regulating target after MAEL promoter methylation. Two are up-regulated genes, and the other twelve genes are down-regulated genes.