A Study On The Association Of The Methylation Status Of HOXA10 And PTEN Gene Promoter Region With Endometriosis

BackgroundEndometriosis(EMs) is one of the most common benign gynecologic disorders, because it has been linked to pelvic pain,multiple operations,and infertility and difficult to cure.It is well known that certain aspects of endometriosis are similar to those of malignant disease.For example,like cancer,endometriosis can be both locally and distantly metastatic;it attaches to other tissues,invades,and damages them.And it is associated with increasing risk of cancer,The ovary is the most common site for development of malignancy in endometriosis,Nowadays the true mechanism for the pathogenesis or therapy of endometriosis is unclear.More and more evidences have indicated that malignant behavior of cancer has already became important morbidity mechanism of endometriosis.Hereditary variation of oncogene and tumor suppressor genes exists in malignant disease,so does endometriosis, especially the methylation of tumor suppressor genes.A number of studys have indicated that methylation of tumor suppressor genes may play an important role in cancer development and progression.Under the majority normal circumstances, status of tumor suppressor genes doesn't show methylation.Convincing evidences for the significance of CpG island or promoter hypermethylation in tumor development stem from the observation that these events are associated with silencing of tumor suppressor genes,eventually it causes cancer.Studies have indicated that HOXA10 gene can decrease invasiveness of cancer cell.This manifested HOXA10 gene is probably tumor suppressor gene.PTEN gene is a universally accepted tumor suppressor gene,which has been observed in a variety of cancers,such as breast cancer,kidney cancer,bladder cancer,melanoma, glioblastoma,and lung cancer,especially the endometrial,ovarian and glioblastoma cancer.The methylation of the CpG island of HOXA10 and PTEN promoter is important mechanism for the pathogenesis of human neoplasms.And this is often associated with metastatic diseases.Aberrant promoter methylation was related to inactivation of the HOXA10 and PTEN gene in ovarian and endometrial cancer. Endometriosis is a kind of benign disease with malignant behavior,and it's associated with ovarian cancer.And then does hypermethylation of HOXA10 and PTEN genes exist in endometriosis?Does the methylation of the CpG islands of HOXA10 and PTEN promoter play an important role in endometriosis? Now it is not clear.So far, the study about these aspects is eliminated,especially about the methylation of PTEN gene in endometriosis.ObjectiveTo investigate the methylation status of the 5'CpG island locating in the promoter region of HOXA10 and PTEN gene in endometriotic tissues and uterine endometrium from women with and without endometriosis in order to study the role of HOXA 10 and PTEN in the pathogenesis and development of the endometriosis.Methods1.Tissue collection:Thirty ectopic endometrium specimens and 20 eutopic endometrium specimens from 30 patients with endometriosis(including 10 cases with endometriosis of stagesⅠ-Ⅱand 20 cases with endometriosis of stagesⅢ-Ⅳ) were used as study group from Sep.2007 to July.2008 in Department of Gynaecology and Obstetrics,Zhujiang Hospital of Southern Medical University,and 25 specimens of endometrium from women without endometriosis were used as controls study in the same period.2.Methods(1) Genomic DNA were extracted from frozen specimens by the method of proteinase K digestion. (2) All samples were detected for the methylation status of the 5'CpG island locating in HOXA10 and PTEN gene promoter region by using methylation-specific PCR technique(MSP).The primers of the 5'CpG island locating in HOXA10 and PTEN gene promoter region were designed according to reference.Set up blank control.(3).Statistical analysis The data were analyzed by using the chi-square test and Fisher's exact test(n＜40 or,when any of the expected frequencies was less than 5), Statistical analysis was performed using SPSS 13.0 software.Results1.The comparison of HOXA10 gene methylation between patients with endometriosis and without endometriosis:In women with endometriosis,presence of HOXA10 promoter methylation was seen in 12 of 30 ectopic endometriums,and 7 of 20 eutopic endometriums.However,there were no methylation occurred in HOXA10 gene of endometrium from 25 women without endometriosis.There were significantly different methylation of HOXA10 gene during three groups(P＜0.05).There were no significantly different methylation of HOXA10 gene not only between in ectopic endometrium and eutopic endometrium of women with endometriosis,but also in eutopic endometrium of women with endometriosis and endometrium of women without endometriosis,While there were significantly different methylation of HOXA10 gene between ectopic endometrium of women with endometriosis and endometrium of women without endometriosis;2.The comparison of PTEN gene methylation between patients with endometriosis and without endometriosis:In women with endometriosis,presence of PTEN gene promoter methylation was seen in 7 of 30 ectopic endometriums,and 2 of 20 eutopic endometriums,.However,there were no methylation occurred in PTEN gene of endometrium from 25 women without endometriosis.There were significantly different methylation of PTEN gene during three groups(P＜0.05). There were no significantly different methylation of PTEN gene not only between in ectopic endometrium and eutopic endometrium of women with endometriosis,but also in eutopic endometrium of women with endometriosis and endometrium of women without endometriosis,While there were significantly different methylation of PTEN gene between ectopic endometrium of women with endometriosis and endometrium of women without endometriosis;3.The comparison of HOXA10 gene methylation between patients with endometriosis inⅠ-Ⅱstage andⅢ-Ⅳstage:In women with endometriosis,presence of HOXA10 promoter methylation was seen in 11 of 20 ectopic endometriums of patients inⅢ-Ⅳstage,and1 of 10 patients inⅠ-Ⅱstage.There were significiantly different methylation of HOXA10 gene in ectopic endometrium between two group patients with endometriosis(inⅠ-Ⅱstage andⅢ-Ⅳstage)(P＜0.05);4.The comparison of PTEN gene methylation between patients with endometriosis inⅠ-Ⅱstage andⅢ-Ⅳstage:In women with endometriosis,presence of PTEN promoter methylation was seen in 6 of 20 ectopic endometriums of patients inⅢ-Ⅳstage,and1 of 10 patients inⅠ-Ⅱstage..There were no significantly different methylation of PTEN gene in ectopic endometrium between two group patients with endometriosis(inⅠ-Ⅱstage andⅢ-Ⅳstage)(P＞0.05).Conclusion1.The methylation of 5'CpG island in the promoter region of HOXA10 and PTEN gene may be involved in occurence of endometriosis;2.The methylation of 5'CpG island in the promoter region of HOXA10 gene may be correlated with the clinical stage of endometriosis and possibly play an important role in the development of endometriosis;3.There may be same biological characteristics about methylation between ectopic endometrium and eutopic endometrium in women with endometriosis.