白细胞介素-4基因甲基化调控与复发性阿弗他溃疡发生机制的相关性研究

Correlation of interleukin-4 gene methylation regulation with the pathogenesis of recurrent aphthous ulcer

目的探讨复发性阿弗他溃疡(recurrent aphthous ulcer,RAU)患者外周血中白细胞介素-4(interleukin-4,IL-4)基因启动子甲基化模式是否发生改变以及环境因素对RAU患者外周血中IL-4基因甲基化水平是否有影响。方法采用调查研究,纳入2018年5月至2019年5月在山西医科大学第一医院口腔门诊就诊并诊断为RAU的患者20例(RAU组),其中女性12例,男性8例,年龄16~35岁。同期在医院体检人员中选取20名与RAU组年龄、性别匹配的健康志愿者作为健康对照组,其中女性11名,男性9名,年龄15~35岁。收集两组受试者外周血样本,采用亚硫酸氢盐处理后测序(bisulfite sequencing PCR,BSP)法检测IL-4启动子甲基化水平,采用实时荧光定量PCR技术检测IL-4 mRNA水平,对所得数据进行统计学分析。结果IL-4基因启动子片段从-1400至-1625 bp中含有10个CPG位点。其中CPG^-1556、CPG^-1483、CPG^-1479的甲基化率以及10个CPG位点的总甲基化率在RAU组[分别为(32.0±19.9)%、(53.0±13.4)%、(46.0±19.8)%及(39.3±12.4)%]均显著高于健康对照组[分别为(20.0±3.2)%、(35.5±12.3)%、(28.0±14.4)%及(32.6±5.8)%](P<0.05)。IL-4 mRNA在RAU组患者外周血中的相对表达量(1.0±0.1)显著低于健康对照组(1.5±0.2)(P<0.01)。RAU组IL-4基因启动子总甲基化率与IL-4 mRNA相对表达量之间存在显著负相关关系(r=-0.494,P<0.05)。在多因素分析中,RAU组吸烟、维生素B12、叶酸与IL-4基因启动子总甲基化率显著相关(P<0.01)。结论RAU患者IL-4基因启动子的高甲基化可能与IL-4基因转录的降低有关;维生素B12、叶酸和吸烟对RAU患者外周血中IL-4基因甲基化水平可能有影响。

Objetive To investigate whether the methylation patterns of the interleukin-4(IL-4)gene promoter changed and whether environmental factors affected the methylation level of IL-4 gene in the peripheral blood of patients with recurrent aphthous ulcer(RAU).Methods Totally 20 patients,who were diagnosed with RAU,were recruited from May 2018 to May 2019 in the Department of Stomatology,First Hospital of Shanxi Medical University in the study(RAU group),including 12 females and 8 males,with mean age of 16-35 years.During the same period,20 healthy volunteers matching the age and gender of the RAU group were selected from the medical personnel of the same hospital as the healty control group,including 11 females and 9 males,with mean age of 15-35 years.Peripheral blood samples of two groups were collected and the methylation levels of the IL-4 promoter were detected by bisulfite sequencing PCR(BSP).The IL-4 promoter methylation level of each sample was analyzed by direct sequencing and the IL-4 mRNA level was detected by real-time quantitative PCR.The data obtained were statistically analyzed.Results The IL-4 gene promoter fragment contained 10 CPG sites from-1400 to-1625 bp.The methylation rates of CPG^-1556,CPG^-1483,CPG^-1479and 10 CPG sites were significantly higher in RAU group[(32.0±19.9)%,(53.0±13.4)%,(46.0±19.8)%and(39.3±12.4)%]than in healthy control group[(20.0±3.2)%,(35.5±12.3)%,(28.0±14.4)%and(32.6±5.8)%],with statistically significant differences(P<0.05).The relative expression of IL-4 mRNA in the peripheral blood of RAU patients(1.0±0.1)was significantly lower than that of the healthy control group(1.5±0.2)(P<0.01).There was a significant negative correlation between the overall methylation rate of IL-4 gene promoter and the relative expression level of IL-4 mRNA in RAU group(r=-0.494,P<0.05).In the multivariate analysis,smoking,vitamin B12 and folic acid in the RAU group were significantly correlated with the overall methylation rate of the IL-4 gene promoter(P<0.01).Conclusions The hypermethylation of IL-4 promoter in RAU patients may be related to the reduction of IL-4 gene transcription.Vitamin B12,folic acid and smoking may affect IL-4 gene methylation in peripheral blood of RAU patients.