Methylation Status of CpG Islands in the Promoter Regions of Signature Genes During Chondrogenesis of Human Synovium-Derived Mesenchymal Stem Cells
Methylation Status of CpG Islands in the Promoter Regions of Signature Genes During Chondrogenesis of Human Synovium-Derived Mesenchymal Stem Cells
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DOI：
10.1002/art.24472
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发表时间：
2009-05-01
影响因子：
--
通讯作者：
Noda, Masaki
中科院分区：
文献类型：
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作者：
Ezura, Yoichi;Sekiya, Ichiro;Noda, Masaki
Objective.人滑膜间充质干细胞（MSCs）可高效分化为成熟软骨细胞。有人认为DNA甲基化是调节人类软骨形成的一种机制;然而，与软骨形成分化相关的基因的甲基化状态尚不清楚。本研究的目的是研究实验性软骨形成过程中人滑膜来源的MSC中CpG甲基化状态，以期在骨关节炎中具有潜在的治疗用途。将人滑膜来源的MSC进行软骨形成团块培养3周。通过亚硫酸氢盐测序分析了分化前后10个候选基因（SOX 9、RUNX 2、CHM 1、FGFR 3、CHAD、MATN 4、SOX 4、GREM 1、GPR 39和SDF 1）启动子中12个区域的甲基化状态。这些基因的表达水平进行了分析，通过实时逆转录聚合酶链反应。甲基化状态也在人类关节软骨进行了检查。亚硫酸氢盐测序分析表明，无论基因的表达水平如何，11个富含CpG的区域中有10个在沉淀培养3周前后的人祖细胞中被低甲基化。在分化后表达增加后，SOX 9、RUNX 2、CHM 1、CHAD和FGFR 3中的甲基化状态始终较低，并且在软骨形成后表达降低后，GREM 1和GPR 39中的甲基化状态较低。一个例外的情况下，一个差异甲基化的CpG丰富的区域是在一个1 kb的上游序列的SDF 1，其表达下降分化。特别地，在沉淀培养3周后，该区域的超甲基化状态降低。软骨细胞表型相关基因的富含CpG的启动子的DNA甲基化水平在人滑膜来源的MSC中的软骨形成期间在很大程度上保持较低。
Objective. Human synovium-derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium-derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis.Methods. Human synovium-derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real-time reverse transcription-polymerase chain reaction. Methylation status was also examined in human articular cartilage.Results. Bisulfite sequencing analysis indicated that 10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture.Conclusion. The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium-derived MSCs.