DNA hypermethylation of extracellular matrix-related genes in human periodontal fibroblasts induced by stimulation for a prolonged period with lipopolysaccharide derived from P. gingivalis
DNA hypermethylation of extracellular matrix-related genes in human periodontal fibroblasts induced by stimulation for a prolonged period with lipopolysaccharide derived from P. gingivalis
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牙龈卟啉单胞菌脂多糖长期刺激诱导人牙周成纤维细胞细胞外基质相关基因DNA高甲基化
DOI：
10.1111/jre.12330
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Yoshihiro Abiko
中科院分区：
文献类型：
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作者：
Rie Takai;Osamu Uehara;Fumiya Harada;Masafumi Utsunomiya;Takatoshi Chujo;Koki Yoshida;Jun Sato;Michiko Nishimura;Itsuo Chiba;Yoshihiro Abiko
目的与背景DNA甲基化与牙周病的关系尚不清楚。牙龈卟啉单胞菌的脂多糖（LPS）参与牙周病的发生发展。我们最近开发了一种LPS感染人牙周成纤维细胞（HPdLFs）的体外模型。在这项研究中，我们检查了用牙龈卟啉单胞菌衍生的LPS刺激长时间的HPdLF中DNA甲基化的全基因组分析。我们注意到细胞外基质（ECM）相关基因的超甲基化，并检查是否超甲基化影响其转录水平。材料和MethodsHPdLFs生长在Dulbecco的改良Eagle培养基中含有10%胎牛血清。重复培养，用牙龈卟啉单胞菌衍生的LPS交替培养3d和不加LPS培养3d，持续1 mo。未处理的样品用作对照。使用人CpG岛微阵列分析DNA。进行定量甲基化特异性聚合酶链反应以确认微阵列数据的再现性。通过定量逆转录-聚合酶链反应（RT-PCR）分析了所选ECM相关基因的mRNA表达水平，发现25个ECM相关基因在启动子区CpG岛发生了高甲基化，其甲基化程度是对照组的4倍。其中，FANK 1、COL 4A 1-A2、12 A1和15 A1、LAMA 5和B1、MMP 25、POMT 1和EMILIN 3等9个ECM相关基因的甲基化导致其mRNA表达显著下调。
Objectives and BackgroundThe involvement of DNA methylation in periodontal disease is not clear. Lipopolysaccharide (LPS) derived fromPorphyromonas gingivalisis involved in the progression of periodontal disease. We recently developed anin vitromodel of LPS infection in human periodontal fibroblast cells (HPdLFs) for a prolonged period. In this study, we examined genome‐wide analysis of DNA methylation in HPdLFs stimulated with LPS derived fromP. gingivalisfor a prolonged period. We noted the hypermethylation of extracellular matrix (ECM)‐related genes and examined whether hypermethylation affected their transcription levels.Material and MethodsHPdLFs were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The culture was repeated, alternating 3 d with LPS derived fromP. gingivalisand 3 d without LPS for 1 mo. Untreated samples were used as controls. DNA was analyzed using the human CpG island microarray. Quantitative methylation‐specific polymerase chain reaction was carried out to confirm reproducibility of the microarray data. The expression levels of mRNA of the selected ECM‐related genes from the data were analyzed by quantitative reverse transcription–polymerase chain reaction.ResultsWe found 25 ECM‐related genes with hypermethylation at the CpG island of the promoter region, which exhibited a fourfold greater hypermethylation than controls. Among these genes, hypermethylation of nine ECM‐related genes, FANK1, COL4A1‐A2, 12A1 and 15A1, LAMA5 and B1, MMP25, POMT1 and EMILIN3, induced a significantly downregulated expression of their mRNA.ConclusionThese results indicate that LPS derived fromP. gingivalismay cause DNA hypermethylation of some ECM‐related genes followed by downregulated expression of their transcriptional levels.