Expression And Significance Of CTLA-4 In Ovrian Cancer

Background and purpose of the studyOvarian cancer is one of the common tumors of the female reproductive system and ranks first in the mortality rate of gynecologic reproductive system malignancies.Since the disease is not specific in early stages and even some patients have no symptoms,it is difficult for most patients to be detected early,resulting in delaying the patient’s disease to relatively advanced stages at the time of diagnosis.Traditional treatments for ovarian cancer include surgery,radiotherapy,chemotherapy,etc.Traditional treatments are effective in early stage,but in advanced and recurrent ovarian cancer there are problems such as high drug resistance and easy recurrence,which cannot achieve the effect of improving the survival rate of patients in advanced stage,and the five-year survival rate of advanced ovarian cancer is very low,less than 30%.How to solve the problems of early diagnosis,late treatment resistance and recurrence of ovarian cancer has become the focus of successful ovarian cancer treatment.The problem facing clinical diagnosis and treatment at this stage is the need to actively search for reliable early diagnosis and molecular markers to improve patient prognosis.Immune checkpoint inhibitor therapy is the fourth tumor treatment modality after surgery,radiotherapy,and chemotherapy,and is mainly used in clinical practice for patients with advanced or recurrent cancer who have failed conventional treatment;although there have been successful clinical cases of Immune Checkpoint Inhibitors(ICIs),their efficacy in gynecologic tumors is limited.The efficacy of ICIs in gynecologic tumors is limited,especially in ovarian cancer patients,where the response rate is only7%-22%;Cytotoxic T Lymphocyte-Associated Antigen-4(CTLA-4)is an important component of the immune checkpoint,and although CTLA-4-related drugs have been approved for clinical use,the number of CTLA-4-related drugs reported so far has been reduced.treatment,but there is limited evidence reported for the clinical use of CTLA-4,and the molecular mechanism is still unclear,and the role in ovarian cancer is less studied in tumor cells still needs to be clearly investigated,and new CTLA-4-related targets still need to be sought to improve clinical efficacy.In summary,this study aimed to investigate the specific expression of CTLA-4 in cancer cells and its role in ovarian carcinogenesis and development,and to provide a richer theoretical basis for the application of CTLA-4 inhibitors in clinical practice.Methods1.Bioinformatics(1)The expression of CTLA-4 in ovarian tissues and tumor tissues was analyzed using the GEPIA database to obtain the expression of CTLA-4 in ovarian cancer.(2)The prognostic value of CTLA-4 in ovarian cancer tissues was predicted by Kaplan-Meier plotter online database.(3)The ovarian cancer GSE dataset was downloaded from the GEO database,and the four microarray datasets were analyzed to obtain data on differential genes(Differentially Expressed Genes,DEGs)using the GEO2 R tool to validate CTLA-4 as a differential gene in ovarian cancer.(4)The raw data were analyzed and plotted by the Venn diagram online tool to screen out the DEGs common to the four datasets,and the absolute value of Log FC was taken to be greater than 1 and P<0.05 as the criterion for screening differential genes.Among them,DEGs with Log FC>0 were down-regulated genes and DEGs with Log FC>0 were up-regulated genes.(5)GO and KEGG functional clustering analysis of differential genes were performed using the DAVID online database.(6)The PPI protein interaction network map of differential genes was obtained by STRING and Cytoscape online database,and the Cyto NCA and Cytohubba modules of Cytoscape database were used to obtain the core target genes of ovarian cancer protein interaction network.2.Tissue level(1)Combined with the experimental purpose,30 patients diagnosed with primary epithelial ovarian cancer and surgically treated in Henan Provincial People’s Hospital from June 2019 to June 2022 were selected for this study,and another 11 patients due to ovarian junctional tumors and 22 patients with benign ovarian tumors were selected and their tissues were taken for the study after signing the informed consent.(2)The expression of CTLA-4 in the tissues was detected by immunohistochemical assay and analyzed whether there was any variability between the tissues of each group.The clinicopathological characteristics of the patients,such as age,presence of lymph node metastasis,tumor size and FIGO stage,were compiled according to the retained cases,and whether there was correlation between CTLA-4 in cancer tissues and each pathological characteristic was analyzed.3.Cellular level(1)To detect CTLA-4 m RNA and protein expression levels in different ovarian cancer cell lines(SKOV3,A2780 cells)and normal ovarian cells(IOSE-80 cells)using fluorescent quantitative PCR(q RT-PCR)and immunoblotting assay(Western-blot);(2)Designing CTLA-4 si RNA and transfecting SKOV3 cells to detect the silencing efficiency of CTLA-4 si RNA;(3)CCK-8 cell activity assay was used to detect the effect of CTLA-4 knockdown on the proliferation ability of ovarian cancer cells.4.Statistical analysisAll data statistics were statistically analyzed using IBM SPSS Statistics 21.0 software.measurement data were expressed as mean ± standard deviation(Mean ± SD),and count data were expressed as percentage(%);t-test was used for comparison of differences between unordered samples,andχ2 test was used for comparison of count data.differences were considered statistically significant when P<0.05.The experimental data involved in the cellular experiments in this paper were subjected to three independent replications,and the differences were marked as "*" when P<0.05;P<0.01,marked as "**";P<0.001,marked as "***".And all the statistical graphs in this were made by Graph Pad Prism 9.0graphing software.Results1.Bioinformatics(1)GEPIA database analysis revealed that CTLA-4 was highly expressed in many human tumor tissues compared with normal tissues,and lowly expressed in normal tissues.The expression of CTLA-4 in ovarian cancer tissues was significantly higher than that in normal ovarian tissues,which was statistically different;(2)Analysis of the K-M Plotter database revealed that patients’ progression-free survival(PFS)and overall survival(OS)were shortened after increased CTLA-4 expression,suggesting a correlation between CTLA-4 expression and patient prognosis;(3)Screening 423 up-regulated genes and 137 down-regulated genes by integrating four datasets(GSE14001,GSE105437,GSE14407,GSE18520),the analysis revealed CTLA-4 as a differentially up-regulated gene in ovarian cancer;(4)Analysis of differential genes revealed negative regulation of T cell proliferation,signal transduction,pathways involved in cancer,glutamatergic synapses,PI3K-Akt signaling pathway,human tumor virus infection,and TGF-β signaling pathway involved in cancer development;(5)The DEGs of the four datasets were analyzed,and the PPI network was constructed for the core genes after Cytoscape analysis of the differential genes,and the important modules in the PPI network were extracted,and the CTLA-4 gene was located among its important modules and was the core target gene.2.Tissue level(1)The expression levels of CTLA-4 in ovarian malignant tumors,ovarian junctional tumors and ovarian benign tumors were statistically different(P<0.05),and the higher the degree of malignancy,the higher the expression level;(2)There was no significant correlation between the expression of CTLA-4 and the stage,lymph node metastasis,age and tumor size of ovarian cancer(P>0.05).3.Cellular level(1)Compared with normal cells(IOSE-80),the expression level of CTLA-4 was increased in ovarian cancer cell lines(SKOV3 and A2780 cells)(P<0.05),with the highest expression level in SKOV3cells;(2)In ovarian cancer cells,high expression of CTLA-4 promoted cell proliferation and was involved in ovarian carcinogenesis and development(P<0.05).Conclusion1.CTLA-4 showed high expression in ovarian epithelial carcinoma tissues;2.CTLA-4 showed high expression in ovarian cancer cells,which could promote cell proliferation and participate in ovarian carcinogenesis and development.