Association Of The Methylation Status Of E-cadherin Promotor With Susceptibility To Endometriosis

Objective: Endometriosis, a main reason for obvious dysmenorrhea,menorrhagia and infertility of reproductive women, is an usual gynecologicaldisease, which influence patient’s life seriously. The mechanism of the illnesshas been unclear yet. The malignant transformation rate of endometrosis isvery low, but it is able to invade and transfer as cancer, which has been provenby clinical research. Therefore, genes about invasion and metastasis may playan important role in the process of endometriosis.E-cadherin, whose chief function is cellular adhesion, is a transmembraneglycoprotein. A reduction in E-cadherin can decrease the intercellularadhesion ability and increase the activity of cells. The reduction of E-cadherinwill enhance the metastasis and invasion ability of cells directly in epithelialtumors. The hypermethylation on promoter sequences is the important way todownregulate E-cadherin in tumors and the expression of its mRNA andprotein will descend too. Study on endometriosis has shown that, thehypermethylation of CDH1gene is detected in ectopic endometrial cell linesof E-cadherin decline, and the E-cadherin expression can recover afterTSA(demethylation) treatment. At the same time, the invading ability of thecells is decreasing.This research will observe the relationship between the methylation statusof E-cadherin gene promotor and the risk of ovarian endometriosis.Consequently, it may offer a theoretical foundation for revealing thepathogenesis of endometriosis and also provide new targets for clinicaltreatment of this disease.Methods:1Specimens collection: case group: fifty ectopic endometriumand eutopic endometrium of patients who underwent surgical treatment forovarian endometriosis(ovarian chocolate cyst) in the Fourth Hospital of Hebei Medical University from August2012to March2013were recruited and theiraverage age is36.74±6.76. All of them were pathologically confirmedendometriosis postoperatively, and there was no history of hormonaltreatments before sugery. Control group: fifty normal endometrium of patientswho underwent total hysterectomy due to cervical intraepithelial neoplasia IIIin the same hospital at the same time, their average age is43.57±5.02. All ofthem had pathologically confirming normal endometrial tissue, withoutmalignant transformation and endometriosis.2Experimental procedure2.1Specimen treatment: Immersed all specimens in RNA later solutionimmediately, and all these should be transferred to a4℃refrigerator storageovernight quickly. Finally, the specimens can be transferred to a-20℃refrigerator storage of preserve.2.2DNA extraction: specimens of50-100mg in control group and casegroup should be grinded for DNA extraction respectively..2.3Detecte the methylation status of E-cadherin gene promotor: theextracted DNA were detected by methylation-specific PCR (MSP) technology,and calculate the methylation rate in case group and control grouprespectively.2.4Verify MSP result: draw methylation positive and negative specimensat random, collect their PCR products for clon sequencing to verify theaccuracy of MSP.2.5RNA extraction: specimens of50-100mg in control group and casegroup should be grinded on ice for RNA extraction respectively.2.6Detect the mRNA expression status of E-cadherin gene: mRNAexpression status was detected by RT-PCR in methylation positive group andmethylation negative group.2.7Detect the E-cadherin expression rate: E-cadherin expression rate wasdetected by flow cytometry in methylation positive group and methylationnegative group.2.8Analysis the experimental data with SPSS software package. Result:1The methylation status of CDH1gene promoter in case group andcontrol groupThe methylation rate of CDH1gene promotor in ectopic endometrium,eutopic endometrium, normal endometrium was32%(16/50),26%(13/50) and8%(4/50) respectively. The methylation rate in ectopic endometrium andeutopic endometrium were both higher than in normalendometrium(χ2=9.00,P=0.003; χ2=5.74,P=0.017). But there was no differencebetween ectopic endometrium and eutopic endometrium (χ2=0.44, P=0.509).2The comparison of mRNA expression rate of CDH1gene inmethylation positive group and methylation negative groupIn methylation positive group, the average mRNA expression rate was0.41±0.20，while that in methylation negative group was0.55±0.07，statisticby t test method, the mRNA expression rate in methylation positive group wassignificantly lower than in methylation negative group (t=-2.50，P=0.023).3The comparison of E-cadherin expression rate in methylation positivegroup and methylation negative groupThe average E-cadherin expression rate was9.58±8.08%in methylationpositive group, while that in methylation negative group was18.72±12.70%.Statistic by t test method, the E-cadherin expression rate in methylationpositive group was significantly lower than in methylation negativegroup(t=-2.35，P=0.027).4The comparison of correlation between mRNA expression rate andE-cadherin expression rate in methylation positive group and methylationnegative groupThe mRNA expression rate and its corresponding protein expression rateof E-cadherin was correlated closely by Pearson Correlation Coefficientstest(r=0.43，P=0.017).Conclusion:The hypermethylation status of CDH1gene promoter in ectopicendometrium of patients with ovarian endometriosis could downregulate the E-cadherin expression through downregulating its mRNA expression, whichcan increase the risk of ovarian endometriosis in women.