Sufaru Irina-Georgeta, Beikircher Gabriel, Weinhaeusel Andreas, Gruber Reinhard
Department of Oral Biology, Medical University of Vienna, Vienna, Austria.
Department of Periodontology, Faculty of Dental Medicine, University of Medicine and Pharmacy "Grigore T. Popa" Iasi, Romania.
J Periodontal Implant Sci. 2017 Apr;47(2):66-76. doi: 10.5051/jpis.2017.47.2.66. Epub 2017 Apr 29.
Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-β1 (TGF-β1).
Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-β1. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification.
We found that 5-aza enhanced TGF-β1-induced interleukin-11 () expression in gingival fibroblasts 2.37-fold (=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 () and NADPH oxidase 4 (). Consistent with this, 5-aza caused demethylation of the gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-β type I receptor kinase inhibitor SB431542 impeded the changes in expression, indicating that the effects of 5-aza require TGF-β signaling. 5-aza moderately increased the expression of TGF-β type II receptor (1.40-fold; =0.009), possibly enhancing the responsiveness of fibroblasts to TGF-β1. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (=0.005) and DNMT3B (=0.002), which are enzymes responsible for gene methylation.
These data suggest that the inhibition of DNA methylation by 5-aza supports TGF-β-induced expression in gingival fibroblasts.
口腔伤口愈合需要牙龈成纤维细胞对局部生长因子作出反应。通过DNA甲基化进行的表观遗传沉默可能会降低牙龈成纤维细胞对局部生长因子的反应性。在本研究中，我们的目的是确定DNA甲基化的抑制是否会使牙龈成纤维细胞对转化生长因子-β1（TGF-β1）敏感。
在使用TGF-β1刺激之前，将牙龈成纤维细胞暴露于5-氮杂-2'-脱氧胞苷（5-aza），一种临床批准的去甲基化剂。使用定量聚合酶链反应（PCR）分析评估基因表达变化。通过甲基化敏感限制酶和PCR扩增检测DNA甲基化。
我们发现5-aza使TGF-β1诱导的牙龈成纤维细胞中白细胞介素-11（IL-11）表达增强2.37倍（P = 0.008）。5-aza对蛋白聚糖4（PRG4）和NADPH氧化酶4（NOX4）的表达没有显著影响。与此一致，5-aza导致牙龈成纤维细胞中通常位于鸟苷（CpG）岛旁边的IL-11基因去甲基化。TGF-βI型受体激酶抑制剂SB431542阻碍了IL-11表达的变化，表明5-aza的作用需要TGF-β信号传导。5-aza适度增加了TGF-βII型受体的表达（1.40倍；P = 0.009），可能增强了成纤维细胞对TGF-β1的反应性。作为反馈反应的一部分，5-aza增加了DNA甲基转移酶1（DNMT1）（P = 0.005）和DNMT3B（P = 0.002）的表达，这两种酶负责基因甲基化。
这些数据表明5-aza对DNA甲基化的抑制支持TGF-β诱导的牙龈成纤维细胞中IL-11的表达。
