RAMP1基因启动子区DNA甲基化状态及MicroRNA与偏头痛相关性

发布时间：2018-06-25 10:37

本文选题：偏头痛 + DNA甲基化　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：背景：偏头痛是一种常见神经系统疾病,临床主要表现为反复发作的单侧中重度搏动性头痛,伴恶心、呕吐、畏光、畏声,活动加重头痛,严重影响患者生活质量、工作能力,被WHO列为第6位失能性疾病。由于复杂的发病机制仍不十分清楚,偏头痛迄今依然缺乏可用于临床的标志物,诊断高度依赖临床症状,严重困扰患病风险评估、临床鉴别诊断,以及预后判断。目前推测表观调节机制可能参与偏头痛的发病过程,如DNA甲基化和miRNA的异常表达,但临床研究很少。大量研究显示降钙素基因相关肽(CGRP)及其受体活性修饰蛋白1 (RAMP1)与偏头痛发病密切相关,然而基因多态性研究并未发现与偏头痛相关。本研究拟从临床角度,探索外周血RAMP1启动子区DNA甲基化水平、循环miRNA (miR-188-5p与miR-382-5p)及CGRP差异性表达是否与偏头痛有关,能否作为偏头痛新的临床标志物。方法：以偏头痛患者及年龄、性别匹配的健康对照作为研究对象,抽取静脉血,分离血清,提取外周血细胞DNA和RNA,以及血清miRNA;亚硫酸盐处理DNA,采用Sequenom公司的Mass ARRAY系统对RAMP1基因启动子区CpG岛进行甲基化定量分析;血清miRNA逆转录后,实时定量PCR检测miR-188-5P和miR-382-5p相对表达;采用实时定量PCR检测外周血CGRP mRNA的表达。分析外周血RAMP1基因启动子区CpG岛甲基化、CGRP mRNA,以及血清miR-188-5P和miR-382-5p和偏头痛相关性。结果：①偏头痛组RAMP1启动子区平均甲基化水平较健康对照组有降低趋势,8.41%±1.92% vs.9.90% ±3.88%,p=0.197：②有偏头痛家族史的患者在(+25,+27,+31,相对于转录起始点)CpG单位的甲基化水平显著高于无偏头痛家族史的患者(13.92%±5.97%vs.8.77% ±6.61%,p=0.034)：③女性患者在(+89,+94,+96)CpG单位的甲基化水平显著低于健康女性(2.18%±1.91% vs.5.85%±5.41%,p=0.02),而在男性患者和男性对照中并没有类似差异;该位点甲基化水平低于3.50%的女性,其罹患偏头痛的风险高于甲基化水平大于3.50%的女性(OR:7.313; 95%CI:1.439-37.164);④有偏头痛家族史患者血清miR-188-5p相对表达量下降4.2倍(p=0.106),发作期miR-188-5p较非发作期相对表达量下降5.54倍(p=0.132),但未达到显著统计学差异;⑤miR-382-5p的相对表达量在偏头痛和对照组之间未见明显差异;⑥外周血CGRP mRNA未见差异性表达。结论：①本研究提示RAMP1基因启动子区DNA甲基化可能参与偏头痛发病,在偏头痛患者中平均甲基化水平有降低趋势,2个CpG单位甲基化水平分别与阳性偏头痛家族史和女性偏头痛相关,其中(+89,+94,+96)CpG单位甲基化可能与女性罹患偏头痛的风险有关;②血清miR-188-5p可能与偏头痛阳性家族史和偏头痛发作有关;③血清miR-382-5p水平和外周血CGRP mRNA表达与偏头痛关联性不密切。上述发现仍需进一步扩大样本量验证,其潜在的分子机制仍需阐明。
[Abstract]:Background: migraine is a common nervous system disease. The main clinical manifestations of migraine are recurrent unilateral moderate and severe pulsatile headache with nausea, vomiting, photophobia, fear of noise, aggravation of headache by activity, which seriously affects the patients' quality of life and ability to work. It was ranked the 6th incapacitated disease by WHO. As the complex pathogenesis is still unclear, migraine is still lack of clinical markers, diagnosis is highly dependent on clinical symptoms, serious trouble disease risk assessment, clinical differential diagnosis, and prognosis judgment. It is speculated that epiregulatory mechanism may be involved in the pathogenesis of migraine, such as DNA methylation and abnormal expression of miRNA, but there are few clinical studies. A large number of studies have shown that calcitonin gene-related peptide (CGRP) and its receptor activity modified protein 1 (RAMP1) are closely related to migraine, but gene polymorphism has not been found to be associated with migraine. The aim of this study was to explore whether the differential expression of circulating miRNA (miR-188-5p and miR-382-5p) and CGRP are related to migraine and whether they can be used as a new clinical marker for migraine. Methods: the patients with migraine and the healthy controls matched by age and sex were used as the study objects. The venous blood was collected and the serum was isolated from the patients with migraine. DNA, RNA and serum miRNAs were extracted from peripheral blood cells, DNA was treated with sulfite, methylation quantitative analysis of CpG island of RAMP1 gene promoter region was performed by mass ARRAY system of Sequenom Company, and miR-188-5P and miR-382-5p relative expressions were detected by real-time quantitative PCR after reverse transcription of miRNA in serum. The expression of CGRP mRNA in peripheral blood was detected by real time quantitative PCR. The relationship between CGRP mRNAs and miR-188-5P, miR-382-5p and migraine in peripheral blood was analyzed. Results the average methylation level of RAMP1 promoter in the migraine group was 8.41% 卤1.92% vs.9.90% 卤3.88% 卤0.197: 2 in patients with family history of migraine. The methylation level of CpG units was significantly higher in patients with a family history of migraine (25,27,31, relative to the starting point of transcription). The methylation level of (89,94,96) CpG units in female patients was significantly lower than that in healthy women (13.92% 卤5.97vs.8.77% 卤6.61p0.034) compared with healthy women (2.18% 卤1.91% vs.5.85% 卤5.41 p0.02), but there was no similar difference between male patients and male controls. The methylation level of the locus was less than 3.50% of women. The risk of migraine was higher than that of women with more than 3.50% methylation level (OR: 7.313; 95 CI: 1.439-37.164). The relative expression of miR-188-5p in patients with family history of migraine decreased 4.2 times (p0.106), and the relative expression of miR-188-5p decreased 5.54 times (p < 0.132) in patients with family history of migraine. Significant statistical difference; There was no significant difference in the relative expression of 5miR-382-5p between migraine and control group. Conclusion this study suggests that DNA methylation in the promoter region of RAMP1 gene may be involved in the pathogenesis of migraine. The average methylation level in migraine patients tended to decrease, and two CpG unit methylation levels were associated with the family history of positive migraine and the female migraine respectively. (89, 94, 96) CpG unit methylation may be associated with the risk of migraine in women. 2Serum miR-188-5p may be related to migraine positive family history and migraine attack. The level of miR-382-5p in serum and CGRP mRNA expression in peripheral blood are not correlated with migraine. These findings need to be further expanded to verify the size of the sample, and its potential molecular mechanism still needs to be clarified.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R747.2

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