Association Of The Methylation Status Of E-cadherin Promoter With Uterine Leiomyoma

Objective: Uterine leiomyoma is a common heath issue of women in their reproductive age, with incidences reported from 20 – 30%. Although leiomyoma is of benign nature, it often causes symptoms such as heavy menstrual bleeding, chronic pelvic pain, recurrent miscarriage and infertility. And the exact pathogenesis of uterine leiomyoma still remains unclear so far. Studies have shown that the occurrence and development of uterine fibroids is associated with some tumor suppressor genes, such as T-cadherin, p53, KLF11, DLEC1, and KRT19 etc. Tumor suppressor gene inactivation or loss of function may be one of the reasons to formation uterine leiomyoma.E-cadherin that is an important tumor suppressor gene and belongs to a member of the family of cell surface glycoproteins plays a prominent role in cell-cell adhesion. It can mediate homogeneity adhesion and maintain the tissue structural integrity. Studies showed that the abnormal expression of Ecadherin plays an important role in the formation and development of many epithelial malignancies,including ovarian cancer, endometrial carcinoma, cervical cancer and sarcomas. In addition, the abnormal expression of Ecadherin also plays an important role in the occurrence and development of some benign invasive disease. Such as: lack of E- cadherin expression is a prerequisite for endometriosis to metastasize. In the studies of uterine fibroids, there are also have reports referred to E- cadherin. Matsuzaki et al. found that E- cadherin have up-regulation in infertile women with uterine leiomyoma.E-cadherin expression loss in cancer has been associated with an E-cadherin gene(CDH1) mutation, allelic gene deletion/loss of heterozygosity or hypermethylation. The accumulation of research suggests that E- cadherin gene(CDH1) promoter hypermethylation has significantly reduced the promoter activity; it is responsible for abnormal E- cadherin gene expression and the occurrence, invasion and metastasis for tumors.This study aims to investigate the association about the promoter region methylation of CDH1 gene and uterine leiomyoma. The meaning of this experiment is to deeply explore the pathogenesis of uterine fibroids from the epigenetic, and also provide new targets for clinical treatment of this disease.Methods: 1 Specimens collection: Endometrium tissues were collected from 102 patients with uterine fibroid who were underwent laparoscopic surgery from January 2014 to August 2014 at Fourth Hospital, Hebei Medical University, China. They are 31 to 58 years old(mean age, 47.67± 4.51 years). In control group, normal endometrium samples were obtained from the women who underwent surgery due to cervical intraepithelial neoplasia III in the same hospital at the same time, whose ages from 26 to 55 years old(mean age, 44.90 ± 8.49 years), All of them had pathologically confirming normal endometrial tissue. All the specimens were collected after patients’ informed consent. 2 Experimental procedures 2.1 Specimen treatment: Immersed all specimens in RNA later solution immediately, and all these should be transferred to a 4℃ refrigerator storage overnight quickly. Finally, the specimens can be transferred to a-20℃ refrigerator storage of preserve. 2.2 DNA extraction: specimens of 50-100 mg in control group and case group should be grinded for DNA extraction respectively. 2.3 Detect the methylation status of E-cadherin gene promoter: the extracted DNA was detected by methylation-specific PCR(MSP) technology, and calculates the methylation rate in case group and control group respectively. 2.4 Detect the E-cadherin protein expression: E-cadherin protein expression was detected by flow cytometry in methylation positive group and methylation negative group. 2.5 Analysis the experimental data with SPSS software package.Result:1 The methylation status of CDH1 gene promoter in case group and control groupThe methylation rate of CDH1 gene promotor in uterine leiomyoma endometrium and normal endometrium was 33.3%(34/102)and 8%(4/50), respectively. The methylation rate of E-cadherin in uterine leiomyoma endometrium was higher than it in normal endometrium(0.001).2 The methylation status of CDH1 gene promoter in proliferating phase and secretory phaseIn case group, the methylation rate of CDH1 gene promoter in proliferative phase and secretory phase was 36.36%(12/33)and 32.35%(23/68), respectively. There was no significant difference between them(P=0.357); in control group, the methylation rate of CDH1 gene promoter in proliferative phase and secretory phase was 6.67%1(1/15) and 9.38%(3/32), There was no significant difference between them, neither(P=0.272).3 The comparison of E-cadherin expression in methylation positive group and methylation negative groupThe average E-cadherin expression was 61.74±17.5 in methylation positive group, while that in methylation negative group was 80.13±40.06. Statistic by nonparametric test method, the E-cadherin expression in methylation positive group was significantly lower than in methylation negative group(Z=-2.045, P=0.041).Conclusion: The abnormal methylation status of CDH1 gene promoter exist in the patients of uterine fibroids, and CDH1 gene promoter methylation may be associated with the loss of E-cadherin expression. This suggests that the CDH1 gene have a certain impact on the occurrence of uterine fibroids.