DNA Hypermethylation in HPDLs Stimulated With LPS for Prolonged Periods

Objectives: DNA methylation is the major mechanism of epigenetic alterations. Although these epigenetic modifications are often observed in malignant and premalignant lesions, the involvement of epigenetic modifications in periodontal disease is not clear. Lipopolysaccharide (LPS) derived from P. gingivalis is involved in the progression of periodontal diseases. In this study, we examined genome-wide analysis of DNA methylation in Human periodontal ligament cells (HPDLs) stimulated with LPS derived from P. gingivalis for prolonged periods. We picked up the hypermethylation of inflammation-related (IR) and extracellular matrix-related (ECMR) genes and examined whether the hypermethylation affected their transcription levels.
Methods: HPDLs were grown in DMEM containing 10% FBS. The culture was repeated alternating 3 days with LPS derived from P. gingivalis (WAKO, 1 µg/ml) and 3 days without LPS for 1month. Untreated samples were used as controls. We extracted DNA and RNA from the samples. DNAs were analyzed by Human CpG Island Microarray (Agilent technology). In order to confirm the reproducibility of the Microarray data, DNA samples were treated with sodium bisulfite and quantitative Methylation Specific PCR　(SYBR® Green).The selected IR and ECMR genes from the data were analyzed in order to show expression of those mRNA by quantitative RT-PCR. Results were compared by Mann-Whitney U test with P-value <0.05 accepted as statistically significant.
Results: Overall, there are 2,910 4X hypermethylated genes and 3,375 hypomethylated genes, respectively. We found 33 IR and 25 ECMR genes with hypermethylation at CpG island. Among these genes, the hypermethylation of 9 IR and 5 ECMR genes induced significantly downregulated expression of their mRNAs.
Conclusions: These results indicate that DNA hypermethylation may be involved in some expression of inflammatory-related and extracellular matrix-related genes in HPDL cells stimulated with LPS derived from P. gingivalis for prolonged periods.