| Objective: Endometriosis represents a common gynecological disease which affects women of reproductive age.Dysmenorrhea,chronic pelvic pain,pelvic mass,sexual intercourse pain and infertility are the predominant symptoms that greatly affect the quality of life of women with endometriosis.Although it is a benign disease,it presents with malignantcharacteristics,such as invasion to surrounding tissues,metastasis to distant locations and recurrence following treatment,So often known as "benign cancer".In recent years,the incidence of EMs has increased significantly,and the age of onset is gradually younger,but its pathogenesis is still unclear.More and more studies have shown that EMs are the result of a combination of environmental and genetic factors.DNA abnormal methylation-mediated epigenetic changes play an important role in the formation and progression of endometriosis.In this study,the human genome methylation status of ovarian EMs in northern China was analyzed by human high-throughput DNA methylation microarray(Human Methylation 450 K Beadchip).The significance of this study was to explore the mechanism of ovarian EMs from epigenetics.Methods: Six patients underwent the eutopic endometrium and ectopic endometrial tissues with ovarian EMs who were hospitalized at the Fourth Hospital of Hebei Medical University,and the normal endometrial tissue of six patients with the same hospital was treated with cervical CINIII.The tissue DNA was first extracted and then modified with bisulfite,and the tissue samples were finally amplified and hybridized using the Illumina Human Methylation 450 K chip.The cluster analysis and significance test were carried out to analyza the different methylation level beteween the endometrial tissues of patients with endometriosis and CINIII.Bioinformatics analysis of the selected differential methylation sites(genes)was performed using Go Miner and the KEGG database to screen out possible DNA abnormalities in the endometriosis.In addition,the m RNA expression differences of GSTM1,IL12 B,SCIN and PIK3R5 were detected by real-time fluorescence quantitative PCR,and the relationship between the expression of GSTM1,IL12 B,SCIN and PIK3R5 was analyzed.Result: 1 Genome-wide analysis of DNA methylation: We use | diffscore | ≥ 20 that P <0.01 for the difference gene,while Delta Data≥0.2 indicates that the degree of methylation is increased,and Delta Data≤-0.2 indicates the degree of methylation decrease.1.1 3788 Cp G sites showed significant differences in the degree of methylation between the eutopic endometrium group and the control endometrium group,which included 3550 hypermethylayion sites and 238 hypomethylation sites.The average methylation level of significant sites is higher in the eutopic endometrium group compared to the control,and the difference was statistically significant(P<0.05).1.2 19999 Cp G sites showed significant differences in the degree of methylation between the eutopic endometrium group and the ectopic endometrium group,which included 11656 hypermethylayion sites and 8343 hypomethylation sites.The average methylation level of significant sites is higher in the eutopic endometrium group compared to the ectopic endometrium group,and the difference was statistically significant(P<0.05).1.3 21277 Cp G sites showed significant differences in the degree of methylation between the ectopic endometrium group and the control endometrium group,which included 10884 hypermethylayion sites and 10393 hypomethylation sites.The average methylation level of significant sites is higher in the ectopic endometrium group compared to the control endometrium group,and the difference was statistically significant(P<0.05).1.4 Unsupervised cluster analysis was consistent with the above results.The methylation difference was small in each group,and the difference in methylation between groups was significant.In summary,the total genome methylation level of the eutopic endometrium group was the highest and the control group was the lowest.2 Analysis of DNA methylation in gene promoter region 2.1 323 genes showed significant differences in the degree of methylation between the eutopic endometrium group and the control endometrium group,which included 317 hypermethylayion genes and 6 hypomethylation genes.The average methylation level of significant genes is higher in the eutopic endometrium group compared to the control,and the difference was statistically significant(P<0.05).2.2 6485 genes showed significant differences in the degree of methylation between the eutopic endometrium group and the ectopic endometrium group,which included 4870 hypermethylayion genes and 1615 hypomethylation genes.The average methylation level of significant genes is higher in the eutopic endometrium group compared to the ectopic endometrium group,and the difference was statistically significant(P<0.05).2.3 4176 genes showed significant differences in the degree of methylation between the ectopic endometrium group and the control endometrium group,which included 2541 hypermethylayion genes and 1634 hypomethylation genes.The average methylation level of significant genes is higher in the ectopic endometrium group compared to the control endometrium group,and the difference was statistically significant(P<0.05).In summary,compared with ectopic endometrial tissue and normal endometrial tissue,the degree of methylation was the highest in eutopic endometrial tissue,which was consistent with the whole genome analysis.2.4 The results of comparison between the eutopic endometrium group and the control group were used as a reference to select a total of 150 genes whose promoter region was statistically significant and significant difference.A total of 41 genes rich in Cp G islands were selected using UCSC and Meth Primer online software.Through the ontology analysis of differential genes,it was found that the difference of DNA methylation in endometriosis mainly involved protein binding,ATP binding,adenine nucleotide binding,catalytic activity,receptor activity,signal transduction activity,transcriptional regulation and so on;cell composition mainly involves cell membrane,plasma membrane,protein extracellular matrix,actin cytoskeleton,synaptic part,biological processes mainly related to biological regulation,cell metabolism,signal to,biosynthesis,gene expression regulation and so on.By analyzing the pathways that may influence the methylation level of genes,it is found that it is mainly focused on the signal pathways such as cancer pathway,Toll-like receptor signaling pathway,JAK-STAT signal pathway and Fcγ receptor signaling pathway.Using the above 41 genes,the eutopic endometrium group and ectopic endometrium group were compared,screening out the significant difference in a total of 17 genes,while the control group and ectopic endometrial group were compared to screen out the significant difference in a total of 17 genes.Finally,the total number of significant genes in the three groups was GSTM1,IL12 B,SCIN,PIK3R5,OR2M1 P,PIPOX,THRSP,SNORA74 B,TCEB1,MYO1 C,SNORD111,ARPP-21,OR9Q2,ZNF738,GFRA4.3 Four genes(GSTM1,IL12 B,SCIN,PIK3R5)with the most significant differences in 15 genes were selected for m RNA level validation.The expression of PIK3R5,SCIN,IL12 B and GSTM1 in ectopic endometrium group was higher than that in eutopic endometrium group and control group.The expression of PIK3R5,SCIN and IL12 B in the control group was higher than that in the eutopic endometrium group.The expression of GSTM1 gene in the eutopic endometrium group was higher than that in the control group.The degree of methylation of each gene is opposite to that of expression and is statistically significant.In summary,DNA methylation levels were significantly inversely correlated with m RNA levels.Conclusion: The detection of DNA methylation status in endometrial tissue and endometrial tissue of endometriosis patients with endometriosis was analyzed by total genome high-throughput methylation technique. 1 The abnormal genomic DNA methylation status in the eutopicendometrium of patients with ovarian EMs may play an important role in the pathogenesis.2 The abnormal genomic DNA methylation status in ectopic endometrium of patients with EMs may be related to the occurrence and development of the disease.3 DNA methylation abnormal genes may be involved in multiple cellular signaling pathways.4 There was a negative correlation between gene expression level and methylation degree.The discovery of these methylation differences helps to reveal the pathogenesis of endometriosis from the molecular level and opens up new directions for the subsequent optimization of drug regimens. |
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