Study On The Mechanism Of GSTM1 In Ovarian Endometriosis

Endometriosis(Endometriosis,EMS)is a disease caused by growing functional endometrial tissue outside the uterine cavity.According to the location of the lesion,it can be divided into ovarian endometriosis endometriosis,peritoneal endometriosis,deep infiltrating endometriosis,and other endometriosis.Ovarian endometriosis is the most common type.Previous studies have shown that there are many theories about the pathogenesis of EMS,but the specific mechanism is still unclear.In recent years,epigenetic regulation and the occurrence of endometriosis have been paid more and more attention.Among them,abnormal DNA methylation may play an important role in the occurrence and development 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.Among them,glutathione S-transferase M1(GSTM1)gene has the most significant difference in methylation level in the three groups of ectopic,eutopic and control intimal tissues.Experiments show that the promoter region of GSTM1 gene in the ectopic endometrial tissue of the ovary is hypomethylated,and its m RNA expression is significantly higher than that of the control endometrial tissue.Through in vitro experimental studies,the increased expression of GSTM1 promotes the proliferation of endometrial glandular epithelial cells and inhibits the apoptosis of endometrial glandular epithelial cells.In order to further clarify the pathogenesis of GSTM1 in endometriosis,transcriptome sequencing was used to detect the transcription level of primary endometrial glandular epithelial cells and glandular epithelial cells transfected with GSTM1 to explore the genes with obvious expression differences affected by GSTM1.This study also used the methylation results of the 450 K chip in the early stage,combined with the data of the expression profile chip(GSE141549)in the Gene Expression Omnibus(GEO),and used the R3.6.1limma package for endometriosis differences.Methylation-differ entially expressed gene screening and research on related pathways.It was found that GSTM1 gene has significant differences in methylation level and expression level in endometriosis ectopic and control endometrial tissues,again suggesting that this gene may play an important role in the occurrence and development of ovarian EMS.This study further verified that GSTM1 gene polymorphism is associated with ovarian EMS,and detected the expression of GSTM1 gene in patients with different genotypes by RT-q PCR,and analyzed the relationship between GSTM1 gene polymorphism and ovarian EMS-related primary infertility.Part one: Screening of downstream target genes after GSTM1 overexpression in endometrial glandular epithelial cellsObjective: To analyze the differentially expressed genes of GSTM1 overexpressed primary endometrial epithelial cells and control cells by RNA-Seq technology,and to explore the regulatory mechanism of GSTM1 in ovarian EMS.Methods: Culture primary endometrial glandular epithelial cells,and transfer p ENTER-GSTM1 plasmid into endometrial glandular epithelial cells to overexpress GSTM1 in the cells.Transcriptome sequencing was performed on endometrial gland epithelial cells transfected with recombinant plasmid p ENTER-GSTM1 and endometrial gland epithelial cells transfected with empty plasmid,and differentially expressed genes were identified.Three genes(CCL5,SKA1,TRIM27)with obvious differences in sequencing were verified by RT-q PCR.Results: 48 hours after p ENTER-GSTM1 plasmid was transfected into endometrial glandular epithelial cells,the m RNA and protein expression levels of GSTM1 were significantly higher than those in the empty plasmid group(P<0.05).A total of 54 differentially expressed genes were screened by transcriptome sequencing,and RT-q PCR verification showed that the expression results of CCL5,SKA1,and TRIM27 were consistent with the RNA-Seq results..Conclusion: Through transcriptome sequencing,it is speculated that GSTM1 may participate in the occurrence and development of ovarian EMS by affecting the expression of downstream differential genes CCL5,SKA1,TRIM27,etc.Part two: Effect of GSTM1 on the biological behavior of endometrial epithelial cells and mechanismObjective: To investigate the pathogenesis of endometriosis by GSTM1 through the downstream effect gene TRIM27.Methods: The correlation between TRIM27 and GSTM1 in m RNA and protein expression in ovarian EMS tissue was detected by Speedman correlation analysis.the m RNA and protein expression differences of TRIM27 gene in ovarian EMS tissue and controlled endometrial tissue were detected by RT-q PCR and immunologization.CCK-8 assay was used to detect the effect of TRIM27 expression on the proliferation activity of endometrial cells.Flow cell technology detects the effect of increased TRIM27 expression on endometrial apoptosis.Transwell tests the effect of increased TRIM27 expression on endometrial cell migration and invasion ability.Western blot detects the effect of increased TRIM27 expression on the downstream target gene of PI3K/AKT signaling path.Results: TRIM27 and GSTM1 showed a clear correlation in expression in ovarian EMS tissue(P<0.001);CCK-8 results showed that the survival rate of endometrial epithal cells in the p EZ-M98-TRIM27 transfecting group was significantly higher than in the p EZ-M98 transfecting group(P<0.01);Flow cytometic results showed that the number of endometrial epithal apoptosis in the p EZ-M98-TRIM27 transfective group was significantly lower than in the p EZ-M98 transfective group(P=0.03).Transwell experiment results showed that the PEZ-M98-TRIM27 transfection group and PEZ-M98 transfection group underwent migration and invasion experiments,and the differences between the two groups were statistically significant(P=0.001).Western blot results showed that there was no significant change in PI3 K and AKT in the PI3K/AKT signaling path after the TRIM27 gene over-expression,while the expression of P-PI3 K and P-AKT was significantly higher than that of the empty vector group(P<0.05).Conclusion: GSTM1 participates in the PI3K/AKT pathway through the downstream effector gene TRIM27 to regulate the proliferation,migration,invasion and anti-apoptosis of ovarian EMS,providing important ideas for the mechanism of ovarian EMS occurrence and development.Part three: Bioinformatics analysis of GSTM1 expression level in ovarianen dometriosisObjective: To discover methylated-differentially expressed genes(MDEGs)in ovarian endometriosis,and explore related hub genes and potential pathways.Methods: We select the genome-wide DNA methylation chip data of the ectopic endometrium and the control endometrium in the pre-methylation450 K chip in our laboratory and the expression profile chip(GSE141549)data in the GEO database.We use the R3.6.1limma package to perform intrauterine MDEGs screening for membrane ectopic disease,and enrichment analysis using gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.We use the STRING database to perform protein interaction(PPI)analysis on differential genes,and find their core genes in the interaction network.RT-q PCR and Immunohistochemistry was used to detect the m RNA expression of 5 core genes ESR1,KRT19,NR5A1,CYP11A1,GSTM1 in 20 cases of ectopic endometrial tissues of ovarian EMS patients and 20 cases of control female endometrial tissues.Results:The combined screening resulted in 37 hypermethylation-low expression genes(Hyper-LGs)and 66 hypomethylation-high expression genes(Hypo-HGs)related to ovarian endometriosis.Through GO analysis,it is found that Hyper-LGs are mainly related to cell differentiation during embryonic placenta development and embryonic organ development regulation.Hypo-HGs are mainly related to the composition of extracellular structures and the formation of extracellular matrix.Through KEGG analysis,it is found that Hyper-LGs are related to JAK-STAT signaling pathway,prolactin signaling pathway,Staphylococcus aureus infection,and the interaction of viral proteins with cytokines and cytokine receptors.Hypo-HGs are related to vascular smooth muscle contraction,arachidonic acid metabolism,linolenic acid metabolism,linoleic acid metabolism and drug metabolism,cytochrome P450 pathways.Based on the PPI network,by combining the four sorting methods of cyto Hubba,it was identified that ESR1,KRT19,PAX8,PRL,SFN,IL-2Rβ,IL-20 Ra and KRT8 are the core genes of Hyper-LG,while NR5A1,CYP11A1,ME1 and GSTM1 are The core gene of Hypo-HG.The expressions of ESR1,KRT19 genes in Hyper-LG and NR5A1,CYP11A1,GSTM1 genes in Hypo-HG were detected by RT-q PCR and Immunohistochemistry in ectopic endometrial tissues and control endometrial tissues.The results were obvious difference(P<0.05)and were consistent with the results of biometric analysis.Conclusion:The joint analysis of differential methylation-difference expression gene helps to analyze the epigenetic regulatory mechanism of endometriosis.In addition,the GSTM1 gene may play an important role in the development of endometriosis through biotric analysis.Part four: Genetic variation of GSTM1 is associated with patients with ovarian endometriosis and endometriosis-related primary infertilityObjective: To investigate the role of the genetic variant of GSTM1 in the development of ovarian endometriosis and its associated infertility risk.Methods:This case-control study included 564 women with ovarian endometriosis and 576 normal women in the control group in northern China.The polymorphism of GSTM1 was genotyped by polymerase chain reaction(PCR)method.To assess the biological significance of polymorphisms,the level of GSTM1 m RNA expression in patients’ endometrial tissues was detected by real-time quantitative polymerase chain reaction(RT-q PCR).Results:Compared with the positive genotype,the null genotype of GSTM1 was associated with the risk of developing ovarian endometriosis(OR=1.29,95% CI=1.02-1.62).In addition,we found that GSTM1 m RNA expression was present in the endometrial tissue of all patients,but the expression level of patients with positive genotype was nearly 10 times higher than that of patients with negative genotype.Further analysis showed that patients with null genotype also had a significantly higher risk of primary infertility than patients with positive genotypes(OR=1.59,95%CI=1.01-2.49).Conclusion:Our results suggest that the GSTM1 polymorphism is not only related to the genetic susceptibility to ovarian endometriosis,but also a potential molecular marker of primary infertility in patients with ovarian endometriosis.