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. 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 found the hypermethylation of alpha I (XII) collagen (XII-collagen) and fibronectin type III domain (FN) out of the hypermthylations. We confirmed their hypermethylation by methylation-specific PCR and hypermethylation of those genes and examined whether the hypermethylation affected their transcription levels.
Methods:
HPDLs (LONZA) were grown in DMEM containing 10% FBS. The culture was repeated alternating 3 days with LPS derived from P. gingivalis (WAKO, 1µg/ml) and 3 days without LPS for 1-4month. Untreated samples were used as controls.
DNA extracted from the samples 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 expression levels of mRNA were evaluated by RT-PCR. Results were compared by Mann-Whitney U test with P-value <0.05 accepted as statistically significant.
Results:
We found the hypermethylation of XII-collagen and FN by the CpG Island Microarray. The hypermethylations of XII-collagen and FN were confirmed by MSP and quantitative MSP. The expression levels of XII-collagen and FN were significantly lower than the controls.
Conclusion:
These results indicate that decreased expression of XII collagen and FN in HPDLs stimulated with LPS derived from P.gingivalis may be due to their DNA hypermethylation.