Study On The Role Of Promoter Methylation Of The MGRN1 Gene In Response To Chemotherapy And Prognosis Of Ovarian Serous Cystadenocarcinoma Patients

Epithelial ovarian cancer(EOC),a common malignant tumor in the female reproductive tract,is the highest cause of death among gynecological malignancies,of which ovarian serous cystadenocarcinoma is the most common pathological type.Most epithelial ovarian cancer patients are treated with cytoreductive surgery debulking combined with platinum drug chemotherapy.Although the prognosis of patients with epithelial ovarian cancer is improved,the overall survival rate of patients with epithelial ovarian cancer was not significantly improved,and the 5-year survival rate was only about 30%.Primary or acquired chemotherapy resistance is a major obstacle to successful treatment of epithelial ovarian cancer.Therefore,the main goal of epithelial ovarian cancer research is to conquer chemotherapy resistance.The occurrence of chemotherapy resistance is the result of multiple mechanisms,but many potential molecular mechanisms are still unclear.Increasing evidence showed that abnormal DNA methylation is one of the most common molecular mechanisms associated with chemotherapy resistance.In the preliminary experiments,reduced representation bisulfite sequencing(RRBS)technology is used to perform genome-wide methylation in chemotherapy resistant and chemotherapy sensitive tissues of ovarian serous cystadenocarcinoma patients.The results showed that there was a significant difference in gene methylation between chemotherapy resistant and chemotherapy sensitive patients with ovarian serous cystadenocarcinoma.Among them,the methylation level of MGRN1(Mahogunin Ring Finger 1)promoter region in the chemotherapy resistant group was significantly higher than chemotherapy sensitive group.Therefore,this study selected MGRN1 for in depth study to explore the possible mechanism involved in the development of chemotherapy resistance in ovarian serous cystadenocarcinoma.Part one Abnormal methylation of MGRN1 promoter region and chemotherapy resistance in patients with ovarian serous cystadenocarcinomaObjective:In order to further clarify the relationship between abnormal methylation of the MGRN1 promoter region and chemotherapy resistance in patients with ovarian serous cystadenocarcinoma.Methods:1.MALDI-TOF Sequenom Mass ARRAY was used to detect the methylation level of MGRN1 promoter region in tissues of ovarian serous cystadenocarcinoma patients;RT-qPCR and immunohistochemical staining methods were used to detect MGRN1 mRNA and protein expression levels in ovarian serous cystadenocarcinoma tissues;2.Spearman analysis clarified the correlation between abnormal methylation and mRNA expression of MGRN1.Results:1.The methylation of the MGRN1 promoter region in the chemotherapy resistance group of ovarian serous cystadenocarcinoma patients was significantly higher than chemotherapy sensitive group.2.The mRNA expression of MGRN1 in the chemotherapy resistance group of ovarian serous cystadenocarcinoma patients was significantly lower than chemotherapy sensitive group.3.The protein expression of MGRN1 in the chemotherapy resistance group of ovarian serous cystadenocarcinoma patients was significantly lower than chemotherapy sensitive group.4.Spearman analysis showed that the methylation of MGRN1 promoter region was negatively correlated with the mRNA expression.Conclusion:The methylation of MGRN1 promoter region is negatively regulated with MGRN1 expression,which may be involved in the chemother-apy resistance through down-regulating MGRN1 expression.Part two The mechanism of MGRN1 in the chemotherapy resistance of ovarian serous cystadenocarcinomaObjective:In order to clarify the role and mechanism of MGRN1 in the chemotherapy resistance of ovarian serous cystadenocarcinoma,we carried out proliferation and apoptosis assays and preliminary explored the mechanism of MGRN1 in the chemotherapy resistance of ovarian cancer cell.Methods:1.Knock down the expression of MGRN1 in SKOV3 cell line by stable transfection technology,and detect the mRNA expression of MGRN1 by RT-qPCR and Western Bolt method.2.CCK-8 and cell apoptosis experiment were used to observe the cellular response to cisplatin after down regulation of MGRN1 in SKOV3 cell lines.3.RT-qPCR and immunohistochemical staining methods were used to detect HSP70 mRNA and protein expression levels in ovarian serous cystadenocarcinoma tissue;Spearman was used to analyze the relationship between HSP70 mRNA and protein expression levels.The TCGA database was used to verify the correlation analysis of MGRN1 mRNA expression and HSP70 mRNA expression level in ovarian serous cystadenocarcinoma tissues.Results:1.The sensitivity to cisplatin in SKOV3 cells with knockdown MGRN1 was significantly reduced(P<0.05).2.The mRNA and protein expression of HSP70 in the chemotherapy resistance group of ovarian serous cystadenocarcinoma patients was significantly higher than chemotherapy sensitive group;Spearman analysis showed that the mRNA expression of MGRN1 was negatively correlated with the HSP70 mRNA expression.Combined with TCGA database information,there is a significant correlation between MGRN1 and HSP70 mRNA exprssion(r=-0.5,P=1E-13).Conclusion:Down regulation the expression of MGRN1 will decrease the cell sensitivity to cisplatin;MGRN1 may regulate the chemotherapy resistance of ovarian cancer cells by regulating the expression of HSP70.Part three Relationship between abnormal methylation of MGRN1 promoter region and prognosis of patients with ovarian serous cystadenocarcinomaObjective:The relationship between abnormal methylation in MGRN1 promoter region and prognosis of ovarian serous cystadenocarcinoma patients was analyzed based on the clinical data and follow-up data.Methods:1.Kaplan-Meier analysis and Cox proportional hazard model were analyzed the relationship of MGRN1 mRNA expression with ovarian serous cystadenocarcinoma patients'prognosis.2.Kaplan-Meier analysis was analyzed the relationship of HSP70 mRNA expression with ovarian serous cystadenocarcinoma patients' prognosis.The Kaplan-Meier plotter database was used to verify the correlation between the expression of HSP70 mRNA and the overall survival of ovarian serous cystadenocarcinoma patients.Results:1.The hypermethylation and lower expression of MGRN1 were related to the poor prognosis of patients with ovarian serous cystadenocarcinoma(P<0.05).2.The lower mRNA expression of MGRN1 is an independent risk factor predicting progression-free survival and overall survival(HR=2.18,95%CI:1.07-4.11,P=0.028;HR=1.92,95%CI:0.99-3.79,P=0.045).3.The higher mRNA expression of HSP70 is related to the poor prognosis of patients with ovarian serous cystadenocarcinoma(P<0.05);The Kaplan-Meier plotter database was used to analyze the correlation between the HSP70 mRNA expression and the overall survival of 1207 ovarian serous cystadenocarcinoma patients.The results showed that the higher expression of HSP70 was related to the poor prognosis of patients with ovarian serous cystadenocarcinoma(P=0.013).Conclusion:The hypermethylation levels of MGRN1 was related to the poor outcome of patients with ovarian serous cystadenocarcinoma;The lower mRNA expression of MGRN1 was an independent risk factors for predicting progression-free survival and overall survival of patients with ovarian serous cystadenocarcinoma;Hypermethylation of the MGRN1 promoter region may be involved in the poor prognosis of patients with ovarian serous cystadenocarcinoma through down regulation of HSP70 expression.In summary,this study is the first study to explore the relationship between abnormal methylation of the MGRN1 promoter region and chemotherapy resistance in patients with ovarian serous cystadenocarcinoma,providing a new assays basis for DNA methylation regulation genes to participate in the poor prognosis and chemotherapy resistance of ovarian serous cystadenocarcinoma.The MGRN1 study showed that:1)the upstream region of MGRN1 was significantly hypermethylated in the cancer tissues of chemotherapy resistant patients with ovarian serous cystadenocarcinoma,2)the lower expression of MGRN1 due to hypermethylation of the upstream region was associated with clinical outcomes of patients with ovarian serous cystadenocarcinoma,3)knockdown of MGRN1 expression could desensitize SKOV3 ovarian cancer cells to cisplatin,4)knockdown of MGRN1 expression in SKOV3 cells could significantly reduce HSP70 mRNA expression,which significantly correlated with the treatment outcomes in cisplatin-treated cancer patients,5)HSP70 expression in the cancer tissues of chemotherapy resistant patients was significantly higher than that of chemotherapy sensitive patients and was related to the clinical prognosis of patients with ovarian serous cystadenocarcinoma.