Laboratory of Molecular & Cellular Biology, Graduate School of Bioresources, Mie University, Tsu, Japan.
Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Tsu, Japan.
PLoS One. 2019 Sep 12;14(9):e0222188. doi: 10.1371/journal.pone.0222188. eCollection 2019.
Environmental and endogenous factors under genetic predisposition are considered to initiate the human intervertebral disc (IVD) degeneration. DNA methylation is an essential mechanism to ensure cell-specific gene expression for normal development and tissue stability. Aberrant epigenetic alterations play a pivotal role in several diseases, including osteoarthritis. However, epigenetic alternations, including DNA methylation, in IVD degeneration have not been evaluated. The purpose of this study was to comprehensively compare the genome-wide DNA methylation profiles of human IVD tissues, specifically nucleus pulpous (NP) tissues, with early and advanced stages of disc degeneration.
Human NP tissues were used in this study. The samples were divided into two groups: early stage degeneration (n = 8, Pfirrmann's MRI grade: I-III) and advanced stage degeneration (n = 8, grade: IV). Genomic DNA was processed for genome-wide DNA methylation profiling using the Infinium MethylationEPIC BeadChip array. Extraction of raw methylation data, clustering and scatter plot of each group values of each sample were performed using a methylation module in GenomeStudio software. The identification of differentially methylated loci (DMLs) and the Gene Ontology (GO) analysis were performed using R software with the ChAMP package.
Unsupervised hierarchical clustering revealed that early and advanced stage degenerated IVD samples segregated into two main clusters by their DNA methylome. A total of 220 DMLs were identified between early and advanced disc degeneration stages. Among these, four loci were hypomethylated and 216 loci were hypermethylated in the advanced disc degeneration stage. The GO enrichment analysis of genes containing DMLs identified two significant GO terms for biological processes, hemophilic cell adhesion and cell-cell adhesion.
We conducted a genome-wide DNA methylation profile comparative study and observed significant differences in DNA methylation profiles between early and advanced stages of human IVD degeneration. These results implicate DNA methylation in the process of human IVD degeneration.
遗传易感性下的环境和内源性因素被认为是引发人类椎间盘（IVD）退变的原因。DNA 甲基化是确保正常发育和组织稳定性的细胞特异性基因表达的重要机制。异常的表观遗传改变在包括骨关节炎在内的多种疾病中起着关键作用。然而，IVD 退变中的表观遗传改变，包括 DNA 甲基化，尚未得到评估。本研究的目的是全面比较人类 IVD 组织（特别是核髓组织）的全基因组 DNA 甲基化谱，特别是在椎间盘退变的早期和晚期阶段。
本研究使用人 NP 组织。将样本分为两组：早期退变组（n=8，Pfirrmann MRI 分级：I-III）和晚期退变组（n=8，分级：IV）。使用 Infinium MethylationEPIC BeadChip 阵列对基因组 DNA 进行全基因组 DNA 甲基化谱分析。使用 GenomeStudio 软件中的甲基化模块对原始甲基化数据进行提取，并对每个样本的每个组值进行聚类和散点图绘制。使用 R 软件和 ChAMP 包对差异甲基化位点（DMLs）和基因本体论（GO）分析进行鉴定。
无监督层次聚类显示，早期和晚期退变的 IVD 样本根据其 DNA 甲基组分为两个主要聚类。在早期和晚期椎间盘退变阶段之间鉴定出 220 个 DML。其中，四个位点在晚期椎间盘退变阶段呈低甲基化，216 个位点呈高甲基化。对包含 DML 的基因进行 GO 富集分析，确定了两个显著的生物学过程 GO 术语，即血友病细胞黏附和细胞间黏附。
我们进行了全基因组 DNA 甲基化谱比较研究，观察到人类 IVD 退变的早期和晚期阶段 DNA 甲基化谱存在显著差异。这些结果表明 DNA 甲基化参与了人类 IVD 退变的过程。
