Hypermethylation of Aging-related Genes in HPDL Cells Stimulated With LPS

Objectives: DNA methylation is the major mechanism of epigenetic alterations. Epigenetics is described as changes in pattern of gene expression not involved in the DNA sequence. Little information about epigenetic modifications in periodontal tissues has been shown. Lipopolysaccharide (LPS) derived from P. gingivalis is involved in the progression of periodontal diseases. The present study investigated 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 aging-related 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 1 month. 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 (MSP) and quantitative MSP(SYBR® Green).The selected regulation of cell aging 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, 2X hypermethylated genes were 5,760 genes and hypomethylated genes 6,864 genes, respectively. We found 8 aging-related genes with hypermethylation at CpG island in promoter region. Among these genes, Klotho and CDKN2A affected their transcriptional levels. The hypermethylations of Klotho and CDKN2A were confirmed by MSP and quantitative MSP.
Conclusions: These results indicate that DNA hypermethylation may be involved in decreased expression of aging-related genes in HPDL cells stimulated with LPS derived from P.gingivalis for prolonged periods.