The Role And Mechanism Of GRHL2 In Ovarian Endometriosis

Endometriosis(EMs)is a common gynecological disorder that is caused by growing functional endometrial tissue(glands and stroma)outside the uterine cavity.Ectopic endometriosis involving the ovary is called ovarian endometriosis.Although several theories and hypotheses have been proposed and demonstrated,the exact mechanismof EMS pathogenesis remains unclear.In recent years,it has been confirmed that abnormal gene expression caused by epigenetic changes including DNA methylation plays an important role in the development and progression of EMs.In the early stage of this research group,the Illumina Human Methylation450 K chip was used to analyze and compare the DNA methylation status of the whole genome in the ectopic,eutopic and control endometrial tissues of ovarian EMS patients.The results showed Grainyhead like 2(GRHL2)gene methylation levels were significantly different among the three groups.GRHL2 gene,as a member of the Grainyhead-like transcription factor family,participates in a variety of physiological processes in vivo.In recent years,the role of GRHL2 in tumorigenesis and development has attracted attention.The expression of GRHL2 is different in different tumors and its role is complex.GRHL2 can be used as a tumor suppressor gene to inhibit tumor occurrence by regulating the process of epithelial-mesenchymal transition(EMT)of tumor cells,and as an oncogene to promote the occurrence and progression of tumor by promoting cell proliferation and inhibiting cell apoptosis.Although EMs is a benign disease,it has the same malignant biological behaviors as tumor,such as proliferation,invasion and metastasis.The relationship between GRHL2 and EMs has not been reported.The purpose of this study was to study the role and mechanismof GRHL2 in ovarian EMs.Firstly,DNA methylation level of GRHL2 gene promoter region in endometrial tissues of ovarian EMs patients and control women was detected.Secondly,real-time PCR and immunohistochemistry were used to detect whether there were differences in the expression of GRHL2 gene at mRNA and protein levels.And lastly,to explore the effect of knockdown GRHL2 gene on primary cultured endometrial glandular epithelial cells and its possible role in the pathogenesis of EMs.Part one: The methylation level of GRHL2 gene promoter region in ovarian endometriosisObjective: To analyze the role of methylation level of GRHL2 gene promoter region in the pathogenesis of ovarian EMS.Methods: SequenomMass ARRAY Methylation was used to detect DNA Methylation level of GRHL2 gene promoter region in ectopic endometriumof ovarian EMs patients and normal endometrial in control women.Results: The analysis fromthe MALDI-TOF mass spectrometry showed that the methylation levels of Cp G sites-526,-658,-774,-800,and-892 in the ectopic endometriumof patients with ovarian endometriosis were significantly higher than those in the normal endometriumof controls(Fig.1B).However,the methylation levels of the-696,-722,and-843 Cp G sites were not significantly different between the two groups.Furthermore,the average methylation level of the eight detectable Cp G sites was significantly higher in the ectopic endometriumof patients with ovarian endometriosis than in the normal endometriumof the controls(P<0.05).Conclusion: Abnormal hypermethylation in the promoter region of GRHL2 gene may be associated with ovarian endometriosis.Part two: The expression of GRHL2 in ovarian endometriosisObjective: To investigate the expression of GRHL2 in ectopic endometriumof ovarian EMs patients and normal endometrial of control women.Methods: RT-qPCR and immunohistochemistry were used to detect whether the mRNA and protein levels of GRHL2 gene were different between the two groups.Results: RT-qPCR results showed the mRNA expression level of GRHL2 was lower in the ectopic endometriumthan in the normal endometrium(P<0.001).Spearman’s correlation analysis showed that there was a significant negative connection between GRHL2 mRNA expression and the average methylation levels of the GRHL2 promoter region(r =-0.528,P<0.001).Immunohistochemical results showed that compared with normal endometrial tissues,the protein expression of GRHL2 was lower in ectopic endometrial tissues(P<0.05).Furthermore,immunohistochemical staining showed that GRHL2 was mainly strongly expressed in the nuclei,contrasting with the weak immunostaining in the cytoplasmof epithelial cells within endometrial tissues.Conclusion: GRHL2 expression was decreased in the ectopic endometriumof patients with ovarian endometriosis.Part three: Study on the effect and mechanismof GRHL2 on the biological behavior of endometrial glandular epithelial cellsObjective: To investigate the possible mechanismof GRHL2 in the occurrence and development of ovarian endometriosis.Methods: Primary cultured endometrial gland-epithelial cells were transfected with Si RNA interference technique to knock down the expression of GRHL2.Interference efficiency was detected by RT-qPCR and Western-blot.Transwell chamber assay was used to detect the changes of migration and invasion ability of endometrial glandular epithelial cells after GRHL2 interference.Meanwhile,western-blot assay was used to detect the changes of EMT-related molecules in the transfected cells.Results : Si RNA-GRHL2 was successfully constructed and used on endometrial glandular epithelial cells,and 48 hours later,RT-qPCR and Western-blot results showed that the amount of GRHL2’s mRNA and protein levels was significantly down-regulated(P<0.05).Transwell chamber test results showed that the migration and invasion ability of endometrial glandular epithelial cells were significantly increased after GRHL2 gene knockdown(P<0.05).Western-blot results showed that the expression of ZEB1,N-cadherin and Vimentin in endometrial glandular epithelial cells increased after GRHL2 gene knockdown,while the expression of E-cadherin decreased(P<0.01).Conclusion:Knock down of GRHL2 expression can promote the migration and invasion of endometrial glandular epithelial cells.Knock down of GRHL2 may regulate the migration and invasion of endometrial epithelial cells through ZEB1.