Division of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
Division of Oral Medicine and Pathology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
J Microbiol Immunol Infect. 2014 Jun;47(3):176-81. doi: 10.1016/j.jmii.2012.08.005. Epub 2012 Sep 24.
BACKGROUND/PURPOSE: Epigenetic alterations such as DNA methylation and histone acetylation are described as changes in the pattern of gene expression not involving the DNA sequence. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis has been shown to inhibit osteoblastic cell differentiation. We examined whether DNA hypermethylation was involved in the inhibitory effect of LPS on osteoblastic differentiation of fibroblasts derived from human periodontal ligament (HPDL).
The HPDL cells were incubated with LPS derived from P. gingivalis at a concentration of 10 μg/ml for 24 h. The cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza). Untreated cells were used as a control. Cell viability was determined using cell proliferation reagent. DNA methyltransferase (DNMT1) and runt-related transcription factor 2 (RUNX2) mRNAs were evaluated by quantitative polymerase chain reaction (RT-PCR). Analysis of RUNX2 DNA methylation was performed using quantitative methylation-specific PCR.
The expression level of RUNX2 was significantly lower in the cells stimulated with LPS than the controls. The presence of 5Aza increased the expression of RUNX2 in cells stimulated with LPS. The expression levels of DNMT1 mRNA in the cells stimulated with LPS were significantly higher than in the control. The presence of 5Aza completely abolished the upregulated expression of DNMT1 in cells stimulated with LPS. The methylation of DNA at 0.1 kb and -1.9 kb in the cells stimulated with LPS was significantly higher than the control.
The results indicate that DNA hypermethylation may be involved in the inhibitory effect of LPS on osteoblastic differentiation in HPDL.
背景/目的：表观遗传改变，如 DNA 甲基化和组蛋白乙酰化，被描述为不涉及 DNA 序列的基因表达模式的变化。源自牙龈卟啉单胞菌的脂多糖（LPS）已被证明可抑制成骨细胞的分化。我们研究了 LPS 是否参与了 LPS 对人牙周韧带（HPDL）来源成纤维细胞成骨分化的抑制作用。
将 HPDL 细胞用浓度为 10μg/ml 的牙龈卟啉单胞菌 LPS 孵育 24 小时。用 DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷（5Aza）处理细胞。未处理的细胞作为对照。用细胞增殖试剂测定细胞活力。用定量聚合酶链反应（RT-PCR）评估 DNA 甲基转移酶（DNMT1）和 runt 相关转录因子 2（RUNX2）mRNA。用定量甲基化特异性 PCR 分析 RUNX2 DNA 甲基化。
与对照组相比，LPS 刺激的细胞中 RUNX2 的表达水平明显较低。5Aza 的存在增加了 LPS 刺激细胞中 RUNX2 的表达。LPS 刺激的细胞中 DNMT1 mRNA 的表达水平明显高于对照组。5Aza 的存在完全消除了 LPS 刺激细胞中 DNMT1 的上调表达。LPS 刺激细胞中 0.1 kb 和-1.9 kb 处的 DNA 甲基化明显高于对照组。
结果表明，DNA 高甲基化可能参与了 LPS 对 HPDL 成骨分化的抑制作用。
