| Background:Ovarian cancer is the most difficult gynecological malignancy to detect in early stage and has the highest mortality rate by far.The 5-year survival rate for patients with advanced epithelial ovarian cancer is only 30% due to the lack of effective screening and targeted therapies.Biological clock genes play an important role in the development of tumors.NR1D1 is a ligand-regulated nuclear receptor of the biological clock and a transcription factor capable of regulating a wide range of biological processes.NR1D1 is involved in tumor development and is associated with poor prognosis and chemotherapy sensitivity.The Cancer Genome Atlas(TCGA)database reveal that NR1D1 is highly expressed in normal ovarian tissues and low in ovarian cancer tissues,while its exact mechanism of role in ovarian cancer is not yet clear.Suppressor of cytokine signaling 3(SOCS3)is positively correlated with the prognosis of ovarian cancer,with silencing of SOCS3 expression enhancing the migration and proliferation of ovarian cancer cells,while overexpression does the opposite.TCGA database shows that a positive correlation between NR1D1 and SOCS3 expression in ovarian cancer.Analysis of the JASPAR website reveals the presence of multiple NR1D1 binding sites in the promoter region of SOCS3,indicating that NR1D1 regulates SOCS3 transcription.SOCS3 is a repressor of the Janus kinase(JAK)/Signal converter and activator of transcription 3(STAT3)signaling pathway.The activation of the JAK/STAT3 signaling pathway,which promotes tumor cell proliferation and inhibits apoptosis,is important for the proliferation and apoptosis of tumor cells including epithelial ovarian cancer cells.However,whether NR1D1 inhibits the proliferation of epithelial ovarian cancer through inhibiting the SOCS3/JAK/STAT3 signaling pathway remains to be investigated.Purpose:This study investigated the association between the expression level of NR1D1 and the clinical regression of ovarian cancer patients and its role in ovarian cancer cell lines and experimental animals,analyzed the relationship between the expression of SOCS3 and STAT3 and the clinicopathological parameters of ovarian cancer patients,analyzed the relationship between NR1D1,SOCS3 and JAK/STAT3 signaling pathways through ovarian cancer cell models and animal experiments,and clarified the role of NR1D1 and SOCS3/JAK/STAT3 signaling pathways in the proliferation of ovarian cancer.Methods:1.Cell culture,immunohistochemical staining,q RT-PCR experiments and western blot were performed to detect the expression level of NR1D1 in epithelial ovarian tissues and cell lines and to analyze the relationship between the expression level of NR1D1 and the clinicopathological parameters of epithelial ovarian cancer patients.Factors affecting the prognosis of ovarian cancer patients were analyzed by proportional risk model(cox).2.Ovarian cancer cell models with NR1D1 overexpression plasmid and NR1D1-sh RNA were constructed by overexpression and knockdown of NR1D1.CCK-8 assay,PCNA cell proliferation assay,cell immunofluorescence staining,caspase-3 activity assay,caspase-9 activity assay,flow cytometry and western blot were applied to detect the effect of NR1D1 on proliferation,cell cycle and apoptosis of ovarian cancer cells.3.NR1D1 overexpression ovarian cancer subcutaneous tumor model was established using nude mice,and subcutaneous tumor tissue necrosis and cell apoptosis were detected by HE staining and TUNEL method,and cell proliferation activity was detected by immunofluorescence staining of subcutaneous tumor tissue.4.Cell culture,immunohistochemical staining,q RT-PCR experiments and western blot were performed to detect the expression level of SOCS3 in epithelial ovarian tissues and cell lines and to analyze the relationship between the expression of SOCS3 and STAT3 and the clinicopathological parameters of epithelial ovarian cancer patients.The expression levels of SOCS3 in ovarian cancer cells and epithelial tumors overexpressing and knocking down NR1D1 were measured by q RT-PCR and western blot to clarify the effect of NR1D1 on SOCS3.The expression levels of JAK and STAT3 in two ovarian cancer cell lines and ovarian cancer subcutaneous tumors overexpressing and knocking down NR1D1 were detected by western blot to clarify the role of NR1D1 on the JAK/STAT3 signaling pathway.A co-transfected ovarian cancer cell model with NR1D1 overexpression and SOCS3 knockdown was constructed,and the proliferation,apoptosis and STAT3 expression levels of the co-transfected ovarian cancer cell model were examined by CCK-8,flow cytometry and western blot to clarify the relation between NR1D1,SOCS3 and JAK/STAT3 signaling pathway in ovarian cancer proliferation.Results:1.The expression levels of NR1D1 were significantly lower in epithelial ovarian cancer cells and tissues than that in normal epithelial ovarian cells and fallopian tube tissues.The results of the chi-square test analysis showed that the expression level of NR1D1 was positively correlated with the clinicopathological grade of ovarian cancer patients,but negatively correlated with FIGO stage and lymph node metastasis.The Kaplan-Meier survival curve showed that the 5-year survival rate was significantly higher in patients with high NR1D1 expression than in those with low NR1D1 expression,and the Cox analysis showed that the expression level of NR1D1 was an independent factor affecting the prognosis of ovarian cancer patients.2.CCK-8 assay and PCNA cell proliferation assay showed that the proliferation ability of ovarian cancer cells overexpressing NR1D1 was significantly reduced,while the proliferation ability of ovarian cancer cells knocking down NR1D1 was significantly enhanced by using the constructed NR1D1 overexpression and knockdown NR1D1 epithelial ovarian cancer cell model.Flow cytometric detection of the cell cycle showed that the proportion of G1 stage cells was significantly higher in ovarian cancer cells overexpressing NR1D1,and western blot showed levels of cyclin D and cyclin E significantly reduced.The proportion of G1 stage cells was significantly lower in ovarian cancer cells knocking down NR1D1,and western blot showed levels of cyclin D and cyclin E significantly increased.Flow cytometry showed that the apoptosis rate of ovarian cancer cells overexpressing NR1D1 was significantly increased,and caspase-3 and caspase-9 activities were significantly enhanced,while the apoptosis rate of ovarian cancer cells knocking down NR1D1 was significantly reduced.3.The results of subcutaneous tumor experiments in nude mice showed that the growth rate of subcutaneous tumors in the NR1D1 overexpression group was significantly lower than that of the control group,and the volume and weight of subcutaneous tumors were significantly lower than that of the control group.HE staining showed that there were obvious areas of apoptosis and necrosis in the NR1D1 overexpression group.The TUNEL assay showed more TUNEL-positive cells in the NR1D1 overexpression group.The results of the immunofluorescence assay showed lower Ki-67 levels in the NR1D1 overexpression group.4.The expression levels of SOCS3 were significantly lower in epithelial ovarian cancer cells and tissues than that in normal epithelial ovarian cells and fallopian tube tissues.The results of chi-square analysis showed that the expression of SOCS3 was positively correlated with the clinicopathological grade of ovarian cancer,and negatively correlated with FIGO stage and lymph node metastasis.The expression of p-STAT3 was negatively correlated with histological grade and positively correlated with FIGO stage and lymph node metastasis.The expression level of SOCS3 was significantly increased and the expression levels of p-JAK1,p-JAK2 and p-STAT3 were significantly decreased in ovarian cancer cells overexpressing NR1D1,while the expression level of SOCS3 was significantly decreased and the expression levels of pJAK1,p-JAK2 and p-STAT3 were significantly increased in ovarian cancer cells knocking down NR1D1.The expression level of SOCS3 was significantly increased and the expression levels of p-JAK1,p-JAK2 and p-STAT3 were significantly decreased in subcutaneous tumors overexpressing NR1D1,while the expression levels of t-JAK1,t-JAK2 and t-STAT3 were not significantly changed.The expression levels of p-JAK1,p-JAK2 and p-STAT3 were significantly decreased in two NR1D1 overexpression ovarian cancer cell lines,while the levels of t-JAK1,t-JAK2 and tSTAT3 did not change significantly.The expression levels of p-JAK1,p-JAK2 and pSTAT3 were significantly increased in two NR1D1 knockdown ovarian cancer cell lines,while t-JAK1,t-JAK2 and t-STAT3 expression levels did not change significantly.The expression levels of p-JAK1,p-JAK2 and p-STAT3 were significantly decreased in subcutaneous tumors overexpressing NR1D1,while the expression levels of t-JAK1,t-JAK2 and t-STAT3 did not change significantly.The proliferation ability of ovarian cancer cell co-transfected with NR1D1 overexpression and SOCS3 knockdown was significantly enhanced,the apoptosis rate was significantly reduced,and the level of p-STAT3 was significantly reduced.Conclusion:1.NR1D1 is lowly expressed in epithelial ovarian cancer and correlates with clinicopathological histological grade,FIGO stage and lymph node metastasis of ovarian cancer,and the expression level of NR1D1 is positively correlated with the prognosis of ovarian cancer patients.2.NR1D1 inhibits the activation of the JAK/STAT3 signaling pathway through upregulation of SOCS3 expression,inducing cell cycle arrest and promoting apoptosis,thereby inhibiting ovarian cancer proliferation.3.NR1D1 plays an oncogenic role in ovarian cancer and could be a potential therapeutic target and prognostic indicator for epithelial ovarian cancer. |
