IADR Abstract Archives
TLR9 gene-promoter’s hypomethylation regulates its active transcription in periapical inflammation
Objectives: To assess the methylation pattern of TLR9 at the body gene and promoter region, and its association with its transcriptional regulation in periapical inflammation
Methods: Cross-sectional study. Apical tissues were obtained from apical lesions of endodontic origin (ALEOs) (n=61) and healthy periodontal ligament (HPL) (n=15) from volunteers having indication of tooth extraction. All samples were homogenized, and both total RNA and DNA were extracted. Analysis of TLR9 gene revealed CpG sites within the gene promoter; and three CpG islands on its exon 2 within the gene body. DNA was bisulfited, amplified by PCR using a validated primer sets and their products were sequenced. The results were analyzed by BiQanalyzer. TLR9 mRNA levels were quantified by qPCR
Results: TLR9 expression levels were higher in ALEOs compared to healthy controls (p<0.05). Global methylation % of the TLR9 gene promoter and body-island 1 were lower in ALEOs compared to HPL (p<0.05), while body-islands 2 and 3 tended to be totally methylated independently of the periapical status (p>0.05). Only the promoter’s methylation % was confirmed to influence gene expression (p<0.05). Analysis of CpG single-sites showed that positions -805, -736, -683, -546, -488, -481, -389, -105, -78, -35 and +2086 were hypomethylated in ALEOs compared to HPL (p<0.05), whereas a higher expression of TLR9 was associated with unmethylated -736 and -683 CpG single-sites within the TLR9 gene promoter in apical tissues (p<0.05). The TLR9 gene expression influence the methylation status of -736 and -683 CpG sites from TLR9 gene promoter in apical tissues, after adjusting by smoking and gender (p<0.05)
Conclusions: CpG island 1 from exon 2 and CpG sites within the promoter of the TLR9 gene were hypomethylated in ALEOs compared to HPL. TLR9 gene promoter hypomethylation, specifically at CpG single-sites near CRE-binding, were involved in higher active transcription of TLR9 in ALEOs
Methods: Cross-sectional study. Apical tissues were obtained from apical lesions of endodontic origin (ALEOs) (n=61) and healthy periodontal ligament (HPL) (n=15) from volunteers having indication of tooth extraction. All samples were homogenized, and both total RNA and DNA were extracted. Analysis of TLR9 gene revealed CpG sites within the gene promoter; and three CpG islands on its exon 2 within the gene body. DNA was bisulfited, amplified by PCR using a validated primer sets and their products were sequenced. The results were analyzed by BiQanalyzer. TLR9 mRNA levels were quantified by qPCR
Results: TLR9 expression levels were higher in ALEOs compared to healthy controls (p<0.05). Global methylation % of the TLR9 gene promoter and body-island 1 were lower in ALEOs compared to HPL (p<0.05), while body-islands 2 and 3 tended to be totally methylated independently of the periapical status (p>0.05). Only the promoter’s methylation % was confirmed to influence gene expression (p<0.05). Analysis of CpG single-sites showed that positions -805, -736, -683, -546, -488, -481, -389, -105, -78, -35 and +2086 were hypomethylated in ALEOs compared to HPL (p<0.05), whereas a higher expression of TLR9 was associated with unmethylated -736 and -683 CpG single-sites within the TLR9 gene promoter in apical tissues (p<0.05). The TLR9 gene expression influence the methylation status of -736 and -683 CpG sites from TLR9 gene promoter in apical tissues, after adjusting by smoking and gender (p<0.05)
Conclusions: CpG island 1 from exon 2 and CpG sites within the promoter of the TLR9 gene were hypomethylated in ALEOs compared to HPL. TLR9 gene promoter hypomethylation, specifically at CpG single-sites near CRE-binding, were involved in higher active transcription of TLR9 in ALEOs