Hypermethylation of Inflammation-related genes in HPDL cells stimulated with LPS

Method: HPDLs 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 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 inflammatory response genes and cytokine production 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.

Result:Overall, 4X hypermethylated genes were 2,910 genes and hypomethylated genes 3,375 genes, respectively. We found 33 inflammation-related genes with hypermethylation at CpG island up to -1000. Among these genes, NFATC1, CXCL, IL28A were the 3 most significant genes in the hypermethylation field. The hypermethylation of NFATC1 and IL17 affected their transcriptional levels.

Conclusion: These results indicate that DNA hypermethylation may be involved in some expression of inflammatory-related genes in HPDL cells stimulated with LPS derived from P.gingivalis.