| Objective: Ovarian cancer(OC)is one of the common malignant tumors of female reproductive organs in clinical practice,and the death rate is still the highest among gynecologic malignancies despite the improvement of treatment methods in recent years.The reason for the high mortality rate of ovarian cancer is that the disease is insidious,and most cases are diagnosed at an advanced stage with extensive pelvic and abdominal metastases.Therefore,it is of great theoretical and practical significance to find more effective methods and indicators for early diagnosis,monitoring recurrence and evaluating prognosis.In this study,we constructed ELF3 overexpression and repression expression ovarian cancer cell line models to deeply explore the effects of ELF3 on proliferation,cycle,invasion and apoptosis of ovarian cancer cells.At the same time,to explore how KLF5 regulates the expression and interaction sites of ELF3,and to provide a theoretical basis for the search of biomarkers related to ovarian cancer metastasis.Methods: 1.Firstly,we screened the ovarian cancer tissue microarray information in GEO database and downloaded 6 tissue m RNA expression profile microarray data of high-grade plasma adenocarcinoma combined with clinical information from TCGA database to identify ELF3 was identified as a candidate molecule for the study.Immunohistochemistry was applied to detect the expression of ELF3 in ovarian cancer tissues and normal ovarian tissues,and to analyze the relationship between ELF3 expression levels and patients’ clinicopathological parameters and prognosis.To verify the expression of ELF3 in normal ovarian tissues,ovarian cancer foci and metastases using q RT-PCR and Western Blot.2.To detect the expression of ELF3 in four ovarian cell lines(IOSE80,SKOV3,A2780,OVCAR3)using RT-q PCR and Western Blot.Overexpression and silent expression of ELF3 in ovarian cancer cell lines were constructed.q RT-PCR and Western Blot were performed to detect changes in m RNA and protein levels of ELF3.Changes in proliferation,cycle,apoptosis,invasion and migration ability of ovarian cancer cells were detected before and after differential expression of ELF3 gene using CCK-8,Ed U,scratch,Transwell and flow cytometry.western Blot was used to detect changes in molecules associated with the above biological behaviors.We constructed subcutaneous transplantation tumors in nude mice to investigate the effect of ELF3 overexpression on the formation of subcutaneous transplantation tumors in nude mice.western blot was performed to detect changes in EMT-related molecules before and after differential expression of ELF3.TGF-β1 was used to induce dynamic changes in EMT-MET model of ovarian cancer cell lines,and ELF3 was knocked down during the MET process.using immunofluorescence,cell morphological changes were observed thus tracking the progress of MET and observing the role of ELF3 in MET.western blot detected the passive expression changes of ELF3 under TGF-β1 stimulation.Cell adhesion assay was performed to detect the changes of cell adhesion function after modulation of ELF3.A nude mouse intraperitoneal implantation model was constructed to observe the effect on the formation of intraperitoneal implantation tumors in nude mice after overexpression of ELF3.3.The transcription factor KLF5 upstream of ELF3 was screened using the JASPAR database.q RT-PCR was performed to detect the expression of KLF5 in normal ovarian tissues,ovarian cancer foci and metastases,and the correlation analysis was performed between the expression of ELF3 and KLF5.Chromatin immunoprecipitation(CHIP),luciferase assay and Electrophoretic Mobility Shift Assay(EMSA)were used to further investigate whether KLF5 binds to the promoter region of ELF3.The KLF5 inhibitory expression ovarian cancer cell lines were constructed.q RT-PCR and Western Blot were performed to detect the changes in m RNA and protein levels of KLF5.CCK-8,Transwell and cell adhesion assays were used to detect the changes in proliferation,invasion and adhesion ability of ovarian cancer cells before and after differential expression of KLF5 gene.And phenotype rescue assay was designed to investigate whether KLF5 regulates the changes of cell proliferation,invasion and adhesion ability through ELF3.Results: 1.The preliminary results based on the combined analysis of GEO database microarray,ELF3 sequencing results in TCGA-GTEx,as well as TCGA survival analysis and Cox regression model,demonstrated that ELF3 may be an independent risk factor for ovarian cancer prognosis,and it was hypothesized that ELF3 gene plays an important role in the development of ovarian cancer.The results of immunohistochemical assay suggested that the expression of ELF3 in ovarian cancer tissues was significantly higher than that in normal ovarian tissues,which was consistent with the conclusion of the survival analysis.Combined with the clinicopathological data,ELF3 protein expression was found to be negatively correlated with the overall and progression-free survival rates of patients.Cox regression analysis of the effects of FIGO stage,ELF3 expression,pathological type,differentiation degree and age factors on patients’ postoperative survival time showed that the expression of ELF3 and FIGO stage were statistically significant in both univariate and multifactorial analyses.ELF3 expression in fresh tissue specimens was higher in primary ovarian cancer foci than in normal ovarian tissues,and higher in metastatic foci than in primary ovarian cancer tissues.2.ELF3 was highly expressed in tumor cells relative to normal ovarian cells,where ELF3 expression was relatively low in A2780 cells and relatively high in SKOV3 cells,so subsequent experiments chose to silence ELF3 in A2780 cells,while SKOV3 cell line was selected to overexpress ELF3.compared with the negative control group,CCK-8,EDU and clone formation assays Transwell assay showed that ELF3 overexpression promoted the proliferation of ovarian cancer cells and silencing of ELF3 decreased the proliferation ability of cells.There was no effect on migration and apoptosis after ELF3 modulation.Cell cycle assay revealed that the number of cells in G0/G1 phase was significantly increased and the number of cells in S phase was decreased after silencing ELF3,and there was no significant change in G2/M.Western Blot results suggested that the expression of cyclin D1 and CDK6 was down-regulated after silencing ELF3.The expression of cyclin D1 and CDK6 was significantly increased in ELF3 overexpression cells.The results of constructing subcutaneous transplantation tumors in nude mice suggested that the volume and mass of tumors in the ELF3 overexpression group were significantly higher than those in the control group.To explore the role of ELF3 in the EMT-MET process,there was no difference in E-cadherin and Vimentin protein level expression after overexpression of ELF3;Ecadherin expression was reduced and Vimentin expression level was not different after silencing ELF3.TGF-β1 was used to induce cells in the EMT-MET model,and after72 h treatment,cells were seen to have a long shuttle-shaped mesenchymal cell morphology change,with reduced E-cadherin expression and increased Vimentin expression on the plasma membrane.After stopping TGF-β1 induction for 72 h,most of the transfected control cells were reversibly transformed to epithelial state,while the silenced ELF3-expressing group still maintained the shuttle-shaped mesenchymal cell state with reduced E-cadherin expression and increased Vimentin expression on the plasma membrane.The cell adhesion assay suggested that the adhesion function was reduced after knockdown of ELF3 and enhanced after overexpression of ELF3.In vivo experiments on nude mice with intraperitoneal injection suggested that metastatic foci formed significantly more and larger in the intraperitoneal cavity of overexpressed ELF3 compared with the control group.3.The transcription factor KLF5 of ELF3 was screened in the JASPAR database.the expression of KLF5 was higher in ovarian cancer metastases and cancer foci than in normal ovarian tissues,and was positively correlated with ELF3 expression(r=0.6802,p<0.05).Immunofluorescence suggested that KLF5 was stably expressed in the nucleus in both A2780 cell line and SKOV3.CHIP experiments demonstrated that KLF5 could bind the promoter region of ELF3.The results of luciferase and EMSA suggested that KLF5 protein and ELF3 promoter interacted with each other.western blot and q RT-PCR results suggested that silencing of KLF5 was successful in the construction of stable transient cell lines,and the protein and m RNA levels of ELF3 were significantly reduced after silencing KLF5 expression.The proliferation ability,invasion ability and adhesion ability of ovarian cancer cells were reduced to different degrees after KLF5 silencing.The results of the rescue assay suggested that transient transfer of si KLF5 in cells stably overexpressing ELF3 partially eliminated the promotion of cell proliferation,adhesion and invasion after overexpression of ELF3;transient transfer of overexpressed ELF3 plasmids in stably transferred silenced KLF5 cell lines significantly rescued the proliferation,adhesion and invasion defects in the absence of KLF5.Conclusions: 1.ELF3 expression was higher in ovarian cancer metastases than in primary foci and significantly higher in both than in normal ovarian tissues.patients with high ELF3 expression had a worse prognosis and was an independent risk factor for the prognosis of ovarian epithelial malignancies.2.ELF3 promotes ovarian cancer cell proliferation,G0/G1 phase transformation,and ovarian cancer cell invasion.ELF3 is also essential for maintaining the epithelial ELF3 is also an essential molecule for maintaining the epithelial state of ovarian cells,and can improve the adhesion and metastatic colonization of ovarian cancer cells.3.The transcription factor KLF5 is highly expressed in ovarian cancer tissues and regulates the expression of ELF3 at the transcriptional level,and promotes the proliferation,adhesion and invasion of ovarian cancer cells. |
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