DNA 甲基化调控骨关节炎软骨细胞中的 Sirtuin 1 表达。

DNA methylation regulates Sirtuin 1 expression in osteoarthritic chondrocytes.

机构信息

Laboratory of Cytogenetics and Molecular Genetics, Faculty of Medicine, University of Thessaly, Larissa, Greece.

Department of Biology, Faculty of Medicine, University of Thessaly, Larissa, Greece.

出版信息

Adv Med Sci. 2023 Mar;68(1):101-110. doi: 10.1016/j.advms.2023.02.002. Epub 2023 Mar 11.

DOI:10.1016/j.advms.2023.02.002

PMID:36913826

Abstract

PURPOSE

Sirtuin 1 (SIRT1) comprises a major anti-aging longevity factor with multiple protective effects on chondrocyte homeostasis. Previous studies have reported that downregulation of SIRT1 is linked to osteoarthritis (OA) progression. In this study, we aimed to investigate the role of DNA methylation on SIRT1 expression regulation and deacetylase activity in human OA chondrocytes.

MATERIALS AND METHODS

Methylation status of SIRT1 promoter was analyzed in normal and OA chondrocytes using bisulfite sequencing analysis. CCAAT/enhancer binding protein alpha (C/EBPα) binding to SIRT1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Subsequently, C/EBPα's interaction with SIRT1 promoter and SIRT1 expression levels were evaluated after treatment of OA chondrocytes with 5-Aza-2'-Deoxycytidine (5-AzadC). Acetylation and nuclear levels of nuclear factor kappa-B p65 subunit (NF-κΒp65) and expression levels of selected OA-related inflammatory mediators, interleukin 1β (IL-1β) and IL-6 and catabolic genes (metalloproteinase-1 (MMP-1) and MMP-9) were evaluated in 5-AzadC-treated OA chondrocytes with or without subsequent transfection with siRNA against SIRT1.

RESULTS

Hypermethylation of specific CpG dinucleotides on SIRT1 promoter was associated with downregulation of SIRT1 expression in OA chondrocytes. Moreover, we found decreased binding affinity of C/EBPα on the hypermethylated SIRT1 promoter. 5-AzadC treatment restored C/EBPα's transcriptional activity inducing SIRT1 upregulation in OA chondrocytes. Deacetylation of NF-κΒp65 in 5-AzadC-treated OA chondrocytes was prevented by siSIRT1 transfection. Similarly, 5-AzadC-treated OA chondrocytes exhibited decreased expression of IL-1β, IL-6, MMP-1 and MMP-9 which was reversed following 5-AzadC/siSIRT1 treatment.

CONCLUSIONS

Our results suggest the impact of DNA methylation on SIRT1 suppression in OA chondrocytes contributing to OA pathogenesis.

摘要

目的

Sirtuin 1（SIRT1）是一种主要的抗衰老长寿因子，对软骨细胞的稳态具有多种保护作用。先前的研究表明，SIRT1 的下调与骨关节炎（OA）的进展有关。在这项研究中，我们旨在研究 DNA 甲基化对人 OA 软骨细胞中 SIRT1 表达调控和去乙酰化酶活性的作用。

材料和方法

采用亚硫酸氢盐测序分析检测正常和 OA 软骨细胞中 SIRT1 启动子的甲基化状态。用染色质免疫沉淀（ChIP）检测 CCAAT/增强子结合蛋白α（C/EBPα）与 SIRT1 启动子的结合。随后，用 5-氮杂-2'-脱氧胞苷（5-AzadC）处理 OA 软骨细胞后，评估 C/EBPα 与 SIRT1 启动子的相互作用和 SIRT1 的表达水平。用 5-AzadC 处理 OA 软骨细胞后，评估核因子 kappa-B p65 亚基（NF-κΒp65）的乙酰化和核水平以及选定的 OA 相关炎症介质白细胞介素 1β（IL-1β）和白细胞介素 6（IL-6）和分解代谢基因（金属蛋白酶-1（MMP-1）和 MMP-9）的表达水平，并用 SIRT1 的 siRNA 转染后进行评估。

结果

SIRT1 启动子上特定 CpG 二核苷酸的高甲基化与 OA 软骨细胞中 SIRT1 表达的下调有关。此外，我们发现 C/EBPα 对高甲基化 SIRT1 启动子的结合亲和力降低。5-AzadC 处理可恢复 C/EBPα 的转录活性，诱导 OA 软骨细胞中 SIRT1 的上调。siSIRT1 转染可防止 5-AzadC 处理的 OA 软骨细胞中 NF-κΒp65 的去乙酰化。同样，5-AzadC 处理的 OA 软骨细胞中 IL-1β、IL-6、MMP-1 和 MMP-9 的表达降低，而在 5-AzadC/siSIRT1 处理后则逆转。