Department of General-, Visceral-, Vascular- and Pediatric Surgery, University of Saarland Medical Center, Homburg 66421, Saar, Germany.
Department of Genetics and Epigenetics, Saarland University, Saarbrücken 66123, Germany.
Aging (Albany NY). 2021 Feb 5;13(4):4831-4849. doi: 10.18632/aging.202621.
The process of aging has been associated with differential patterns of DNA methylation which relate to changes in gene expression. Hence, we aimed to identify genes with significant age-related methylation differences and to study their mRNA and protein expression profile. Genome-wide DNA methylation analysis was performed with the Illumina Infinium Methylation EPIC BeadChip Microarray on bisulfite-converted DNA prepared from monocytes derived from young (average age: 23.8 yrs) and old (average age: 81.5 yrs) volunteers that are separated by at least 50 years of age difference, n=4, respectively. Differentially methylated CpG sites were assigned to the associated genes and validated by deep sequencing analysis (n=20). Demonstrating an age-associated significant increase of methylation in the promoter region (p=1x10), Homeobox A5 (HOXA5), also known to activate p53, emerged as an interesting candidate for further expression analyses by Realtime PCR, ELISA and Western Blot Analysis (n=30, respectively). Consistent with its hypermethylation we observed significant mRNA downregulation (p=0.0053) correlating with significant downregulation (p=0.0431) in the old cohort. Moreover, we observed a significant change in HOXA5 protein expression (p=3x10) negatively correlating with age and promoter methylation thus qualifying HOXA5 for an eligible p53-related aging marker.
衰老过程与 DNA 甲基化的差异模式有关，这些模式与基因表达的变化有关。因此，我们旨在鉴定与年龄相关的甲基化差异具有显著意义的基因，并研究它们的 mRNA 和蛋白质表达谱。使用 Illumina Infinium Methylation EPIC BeadChip Microarray 对来自年轻（平均年龄：23.8 岁）和老年（平均年龄：81.5 岁）志愿者的单核细胞进行全基因组 DNA 甲基化分析，这些志愿者之间至少相差 50 岁，分别有 n=4。差异甲基化 CpG 位点被分配给相关基因，并通过深度测序分析进行验证（n=20）。在启动子区域（p=1x10）中观察到与年龄相关的甲基化显著增加，同源盒 A5（HOXA5），也被证明可以激活 p53，作为进一步表达分析的候选基因，通过 Realtime PCR、ELISA 和 Western Blot 分析（n=30，分别）。与它的高甲基化一致，我们观察到在老年队列中 mRNA 显著下调（p=0.0053），与显著下调（p=0.0431）相关。此外，我们观察到 HOXA5 蛋白表达的显著变化（p=3x10）与年龄和启动子甲基化呈负相关，从而使 HOXA5 有资格成为与 p53 相关的衰老标志物。
