The Expression And Epigenetic Regulation Of Syncytin-1in Endometriosis

Objective Our aim is to investigate the expression of syncytin-1in endometriosis and the endometrium of endometriosis women. Through detecting the global methylation level of genome DNA, we ascertained the relationship between global methylation and syncytin-1expression, and investigated the role of global methylation in the regulation of syncytin-1expression. To study the relationship between the CpG islands methylation of promoter region and the expression of syncytin-1,investigate the role of the CpG islands methylation of promoter region in the expression of syncytin-1. To study the relationship between DNMT3b isoforms and the expression of syncytin-1, and investigate the relativity between DNMT3b isoforms and CpG island methylation of syncytin-1promoter region, explore the role of DNMT3b isoforms in the regulation of syncytin-1expression.Methods The RNA was isolated from the endometiosis and endometrium tissue, and RT-PCR was performed to obtain the cDNA. Real-time PCR and immunohistochemistrical staining were applied to detect the expression of syncytin-1at mRNA level and protein level respectively in endometriosis and the endometrium of endometriosis women. The endometrium of endometriosis women was used as the control group. LINE-1was chosen as the representative of global methylation, and PCR for LINE-1was done using the bisulfite converted DNA as template. Cobra assay was introduced to study the methylation status of LINE-1.5-Mc staining was also performed to detect the global methylation status. Nested PCR for syncytin-1promoter region was performed using the bisulfite converted DNA, and agarose gel electrophoresis was run to separate the PCR products. PCR product for syncytin-1was purified and ligated into specific vector, and incubated for14h in LB in37℃. Miniprep was done to retrieve the DNA, and DNA sequencing was done. Cobra assay was also employed to investigate the methylation status of syncytin-1promoter region. RNA was isolated from the endometriosis and endometrium tissue, and RT-PCR was done to obtain the cDNA. Real-time PCR for DNMT3b isoforms was performed using the cDNA.Results The mRNA level of syncytin-1in endometriosis was nearly as1.9times as in the control group. Immunostaining result revealed that, the protein level of syncytin-1in endometriosis was higher than that in the control group. The Cobra assay showed that, the methylation proportion of LINE-1was74%,75%in endometriosis and control group respectively. The5-Mc immunostaining also indicated that there was no significant difference between the two groups. The sequencing data showed that, the proportion of CpG island methylation was84%and95%in endometriosis and control group respectively. Demethylation happened at the second CpG site most frequently. Cobra assay revealed that, the proportion of CpG island methylation was51%,65%in endometriosis and control group respectively. Compared to control group, the DNMT3b3was decreased by about40%in endometriosis, and DNMT3b7was increased about100%. The level of DNMT3b6was too low to be detected, and the expression of other isoforms was not significantly changed.Conclusion Syncytin-1was expressed both in endometriosis and the control group, and the mRNA level and protein level of syncytin-1in endometriosis were higher than that in the control group. There was no significant difference in global methylation between endometriosis and control group, and this may suggest that global methylation was not involved in the regulation of syncytin-1expression. Compared to control group, the CpG island methylation status of syncytin-1promoter region was lower in endometriosis, and demethylation of CpG islands of syncytin-1promoter region could upregulate the expression of syncytin-1. The downregulation of DNMT3b3and upregulation of DNMT3b7may cooperate to mediate the expression of syncytin-1.