谷胱甘肽硫转移酶mu1基因启动子区异常甲基化与卵巢子宫内膜异位症的关系研究

Relationship between Abnormal Methylation of GSTM1 Promoter Region and Ovarian Endometriosis

背景许多证据表明与正常内膜相比,子宫内膜异位症(EMT)患者的在位内膜和异位内膜中存在许多差异甲基化基因,可能在疾病发生中发挥着重要作用。目的探讨谷胱甘肽硫转移酶mu1(GSTM1)基因启动子区异常甲基化与EMT的关系。方法选取2013年9月—2015年12月于河北医科大学第四医院妇科行腹腔镜手术治疗并经病理检查证实为卵巢EMT的患者65例,另选取同期在该院因宫颈上皮内瘤变(CIN)Ⅲ级行全子宫切除术的患者(对照者)53例,术中无菌采集卵巢EMT患者的在位内膜和异位内膜以及对照者的子宫内膜(对照内膜)。采用焦磷酸测序和实时定量荧光聚合酶链式反应(RT-qPCR)检测内膜组织GSTM1基因启动子区片段一(包含-116、-111、-104、-98 CpG位点)、片段二(包含-40、-23、-16 CpG位点)平均甲基化水平和mRNA表达水平,并分析二者之间的相关性。结果在位内膜和异位内膜片段一平均甲基化水平低于对照内膜(H=2.588,P=0.046;H=6.496,P<0.001),异位内膜片段一平均甲基化水平低于在位内膜(H=4.213,P<0.001)。异位内膜片段二平均甲基化水平低于在位内膜和对照内膜(H=7.693,P<0.001;H=8.257,P<0.001),在位内膜与对照内膜片段二平均甲基化水平比较,差异无统计学意义(H=0.682,P=0.504)。在位内膜和异位内膜mRNA表达水平高于对照内膜(H=6.994,P=0.011;H=3.414,P<0.001),异位内膜mRNA表达水平高于在位内膜(H=3.846,P<0.001)。GSTM1 mRNA表达水平与其启动子区片段一、片段二平均甲基化水平均呈负相关(rs=-0.61,P<0.001;rs=-0.52,P<0.001)。结论卵巢EMT患者的在位内膜和异位内膜呈现GSTM1基因启动子区异常低甲基化和mRNA高表达,提示GSTM1启动子区异常甲基化可能在卵巢EMT的发生和发展中起重要作用。

Background Evidence has shown that many differentially methylated genes exist in eutopic and ectopic endometrium of endometriosis(EMT)patients,which may be closely associated with the development of EMT.Objective To investigate the relationship between abnormal methylation of GSTM1 promoter region and ovarian EMT.Methods Participants were selected from Department of Gynecology,the Fourth Hospital of Hebei Medical University from September 2013 to December 2015,including 65 with pathologically confirmed ovarian EMT using laparoscopic surgical treatment,and a control group of 53 with CINⅢusing total hysterectomy.The eutopic and ectopic endometriotic tissues of ovarian EMT patients and endometrial tissues of controls were collected aseptically during the operation.Pyrosequencing and RT-qPCR were employed to detect the average methylation levels in the first fragment(including-116,-111,-104,-98 CpG sites)and second fragment(including-40,-23,-16 CpG sites)of GSTM1 promoter region and mRNA expression levels,and their correlations were analyzed.Results The average methylation level of the first fragment in the eutopic or ectopic endometrium was significantly lower than that in the control endometrium(H=2.588,P=0.046;H=6.496,P<0.001).The average methylation level of the first fragment in the ectopic endometrium was significantly lower than that in the eutopic endometrium(H=4.213,P<0.001).The average methylation level of the second fragment in the ectopic endometrium was significantly lower than that in the eutopic endometrium or control endometrium(H=7.693,P<0.001;H=8.257,P<0.001),but there was no significant difference between the eutopic endometrium and control endometrium(H=0.682,P=0.504).The average mRNA expression level in the eutopic or ectopic endometrium was significantly higher than that in the control endometrium(H=6.994,P=0.011;H=3.414,P<0.001).Moreover,the average mRNA expression level in the ectopic endometrium was significantly higher than that in the eutopic endometrium(H=3.846,P<0.001).The expression level of GSTM1 mRNA was significantly negatively correlated with the average methylation level of the first or second fragment in GSTM1 promoter region(rs=-0.61,P<0.001;rs=-0.52,P<0.001).Conclusion There were abnormal hypomethylation and high mRNA expression in eutopic and ectopic endometrium in GSTM1 promoter region in patients with ovarian EMT,suggesting that abnormal hypomethylation of GSTM1 promoter region may play an important role in the development of ovarian EMT.