| Objective:Ovarian cancer is the most lethal tumor in gynecology,which is insidious and highly malignant.The main treatment is surgery combined with chemotherapy,progression-free survival(PFS)and overall survival(OS)of patients with ovarian cancer can be prolonged by targeted therapy.However,the 5-year survival rate is still low due to relapse and resistance.Therefore,it is very important to explore the pathogenesis of ovarian cancer and find more effective therapeutic targets.More and more studies have shown that the regulation of ER homeostasis plays an important role in the occurrence and development of malignant tumors.DDRGK1(DDRGK domaincontaining protein 1)is a membrane protein of the endoplasmic reticulum(ER)and plays an important role in the maintenance of ER homeostasis.At present,DDRGK1 has been reported in a few tumors,but it has not been studied in ovarian cancer.The purpose of this study was to investigate the expression of DDRGK1 in ovarian cancer,and its effect on the malignant phenotype and ER homeostasis of ovarian cancer,and to provide a theoretical basis for finding new therapeutic targets for ovarian epithelial malignant tumors.Methods:A total of 125 ovarian tissue samples from a Shenyang Hospital cohort were collected,the expression of DDRGK1 in ovarian cancer,borderline ovarian tumor,benign ovarian tumor and normal ovarian tissue was detected by immunohistochemistry.The correlation between the expression of DDRGK1 and clinicopathological parameters was analyzed.Cox regression analysis was used to analyze the independent prognostic risk factors of ovarian cancer.Gene Expression Profiling Interactive Analysis(GEPIA)and Kaplan-meier plotter database were used to gene expression profiling the relationship between DDRGK1 expression level and the prognosis of ovarian cancer patients.The relationship between the expression level of DDRGK1 and the prognosis of ovarian cancer patients in Shenyang Hospital was verified by survival analysis.Western Blot analysis of DDRGK1 protein expression in four human ovarian cell lines(CAOV3,OVCAR3,ES-2,SKOV3)and human ovarian epithelial cell lines(HOSEpi C)was performed.To investigate whether there is endoplasmic reticulum(ER)stress in human ovarian cancer cell lines,qRT-PCR and Western Blot were used to detect the mRNA and protein expression levels of ER stress markers BIP(immunoglobulin heavy chain binding protein)and CHOP(C/EBP homologous protein).2.The ovarian cancer cell model was established by using OVCAR3 and ES-2 cell lines to express DDRGK1 and inhibit DDRGK1 expression,respectively.The transfection efficiency of the model was detected by qRT-PCR and Western Blot.The proliferation,cell cycle,apoptosis,invasion and migration of ovarian cancer cells were detected by CCK8,flow cytometry,scratch and Transwell methods before and after the differential expression of DDRGK1.Western Blot was used to detect the expression of proteins closely related to cell proliferation,cell cycle and apoptosis before and after the differential expression of DDRGK1.qRT-PCR was used to detect the changes of ER stress markers BIP and CHOP mRNA before and after the differential expression of DDRGK1.To investigate the effect of overexpression of DDRGK1 on proliferation of nude mice subcutaneously transplanted tumor,and to detect the expression of DDRGK1 and Ki-67 by immunohistochemistry.3.Western Blot was used to detect the phosphorylation level of NF-κB signaling pathway and nuclear ectopy of DDRGK1 before and after the differential expression of DDRGK1.The changes of proliferation,cell cycle,apoptosis,invasion and migration of ovarian cancer cells were detected by adding NF-ΚB pathway inhibitor to the overexpression of DDRGK1 and its control group.4.Using PROMO and JASPAR database to screen XBP1(X-box Binding Protein-1),a transcription factor upstream of DDRGK1,the binding of XBP1s(the spliced form of X-box binding protein 1)to the transcriptional regulatory region of DDRGK1 was detected by Chromatin Immunoprecipitation-LRB-Ch IP),the expression of DDRGK1 was detected by Western Blot.The expression of XBP1 s in ovarian tissue was detected by immunohistochemistry,the relationship between the expression of DDRGK1 and the clinicopathological parameters(FIGO stage,pathological type,lymph node metastasis,histological grade)of ovarian cancer was analyzed.The correlation of XBP1 and DDRGK1 expression in ovarian cancer at mRNA level was verified by Cbioportal database.Results: 1.The expression of DDRGK1 was increased in ovarian epithelial malignant tumors.The positive rate of DDRGK1 was correlated with FIGO stage and lymph node metastasis(P<0.05),the survival time of DDRGK1 low expression group was significantly longer than that of DDRGK1 high expression group.The analysis of GEPIA and Kaplan-meier plotter database indicated that the high expression of DDRGK1 was associated with poor prognosis in ovarian cancer patients.The survival analysis of the cohort of patients in Shengjing Hospital suggested that the high expression group of DDRGK1 had a poor prognosis.ER stress exists in human ovarian cancer cell lines OVCAR3 and ES-2.Overexpression of DDRGK1 promoted the migration,invasion and proliferation of human ovarian cancer cell lines OVCAR3 and ES-2,increased the transformation from G1 phase to S phase,and inhibited the apoptosis(all P<0.05)The expressions of PCNA,cyclin D1 and Bcl2 related to proliferation,cell cycle and apoptosis were significantly increased,while Bax protein was down-regulated.Inhibition of DDRGK1 expression had the opposite effect.After overexpression of DDRGK1,the mRNA expression of BIP and CHOP in ER decreased,but the mRNA expression was inhibited.The growth rate,volume and weight of transplanted tumor induced by DDRGK1-overexpressing cells in nude mice were significantly higher than those in the control group,and the expression of Ki-67 in DDRGK1-overexpressing group was also significantly higher than that in the control group.3.After overexpression of DDRGK1,the phosphorylation of P65 in NF-κB pathway was significantly increased,and the nuclear translocation was increased.After inhibition of DDRGK1 expression,the phosphorylation of P65 in NF-κB pathway was significantly decreased,and the nuclear translocation was decreased.Furthermore,PS-341,an inhibitor of NF-κB pathway,was added to the overexpression group of DDRGK1.It was found that PS-341 could significantly inhibit the proliferation,migration,invasion and G1 transformation to S phase of ovarian cancer cells,it promoted apoptosis(P<0.05).The transcription factor XBP1 s can bind to the-905 ～-892 site(AGAGCCACCTC)in the promoter region of DDRGK1.After the expression of XBP1 s was inhibited,the expression of DDRGK1 was significantly decreased.The positive rate of XBP1 s was correlated with the increase of FIGO stage and histological grade(P<0.05),and the survival rate of the patients in the Shengjing Hospital cohort was significantly higher than that in the control group(P<0.05),patients with high expression of XBP1 s had shorter overall survival.Conclusion: 1.DDRGK1 is highly expressed in ovarian cancer tissues and cells.The high expression of DDRGK1 in ovarian cancer tissues is related to FIGO stage and lymph node metastasis.Patients with high expression of DDRGK1 had shorter overall survival.There is endoplasmic reticulum stress in human ovarian cancer cell lines.2.DDRGK1 promotes the transformation of ovarian cancer cells from G1 to S phase,promotes proliferation,migration,invasion,inhibits apoptosis,and maintains the endoplasmic reticulum homeostasis.3.DDRGK1 affects the malignant biological behavior of ovarian cancer by promoting the phosphorylation of NF-κB pathway and nuclear ectopia,and XBP1 s regulates the expression of DDRGK1 by binding to the promoter region of DDRGK1. |
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