Laboratory of Periodontal Biology, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
Faculty of Dentistry, Universidad Andres Bello, Santiago, Chile.
Int Endod J. 2020 Sep;53(9):1229-1237. doi: 10.1111/iej.13329. Epub 2020 Jun 19.
To determine the methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis.
In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n = 17) and symptomatic apical periodontitis (SAP) (n = 17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P < 0.05).
TLR2 depicted a hypomethylated DNA profile at the CpG island in SAP when compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P < 0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P < 0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P < 0.05).
TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.
确定 TLR2 基因启动子的甲基化模式及其与有症状和无症状根尖周炎中根尖周炎症和血管生成反应的转录调节之间的关系。
在这项病例对照研究中，从计划拔牙的无症状根尖周炎（AAP）（n=17）和有症状根尖周炎（SAP）（n=17）志愿者的根尖病变中提取总 RNA 和 DNA。对 DNA 进行亚硫酸氢盐处理，通过 qPCR 扩增 TLR2 基因内 CpG 岛的一段，然后对产物进行测序。此外，通过 qPCR 分析 TLR2、TLR4、IL-6、IL-12、TNFalpha、IL-23、IL-10、TGFbeta、VEGFA 和 CDH5 的 mRNA 表达。使用卡方检验、Mann-Whitney 检验或非配对 t 检验和 Spearman 相关分析对数据进行分析；使用多元线性回归进行变量调整（P<0.05）。
与 AAP 相比，SAP 中 TLR2 在 CpG 岛上呈现低甲基化 DNA 图谱，同时 TLR2 表达上调，促炎细胞因子 IL-6 和 IL-23 以及血管生成标记物 CDH5 也上调（P<0.05）。TLR2 甲基化百分比与根尖周炎中 IL-23 和 CDH5 的 mRNA 水平呈负相关。在 SAP 中，与 AAP 相比，TLR2 启动子中 NFκB 结合附近的单个 CpG 二核苷酸 -8 和 -10 的低甲基化频率降低（P<0.05）。最后，与甲基化相比，未甲基化的 -10 和 -8 单个位点显示出 IL-23、IL-10 和 CDH5 转录物的上调（P<0.05）。
TLR2 基因启动子的低甲基化与炎症加剧时促炎细胞因子和血管生成标记物的转录活性有关。此外，NFκB 结合附近的未甲基化单个位点参与了 IL-23、IL-10 和 CDH5 的活跃转录。
