Expression Of STAT3 And Grim19 In Epithelial Ovarian Cancer And Their Effects On The Biological Function Of Ovarian Cancer Cells

The characteristics of ovarian cancer were these of high mortality rate,concealed onset,rapid disease progression,recurrence and metastasis.These unique disease characteristics bring many problems in clinical treatment of ovarian cancer,and caused poor prognosis of patients.In current studies,STAT3 is a key factor playing a significant role in tumor disease progression and tumor microenvironment.It is constitutively activated in various cancers and affects many biological progresses such as proliferation,apoptosis,tumor microenvironment,and tumor stem cell generation.However,the mechanism and effective targets of STAT3 need further study.Grim19 is a tumor suppressor gene discovered in recent studies.As a constituent subunit of mitochondrial complex I,it participates in energy metabolism.At the same time,studies have shown that Grim19 has a specific effect on STAT3,and the two genes interact with each other,but the interacting mechanism is not clear.In this study,bioinformatics methods were used to screen genes and action signal pathways related to STAT3 and Grim19,and histological experimental methods were used to study the expression,location and the relationship with clinicopathological factors of STAT3 and Grim19 in epithelial ovarian cancer,and cell experiments in vitro were used to explore the effects of STAT3 and Grim19 on the function of ovarian cancer cells.Chapter 1.Using Bioinformatical Method Predicts the Related Genes and Possible Signal Pathways of STAT3 and Grim19Objective Using the bioinformatical method analyses the basic information,and screens the related gens and possible signal pathways.Methods Use NCBI,UCSC,Uni Prot database to analyses the basic information of STAT3 and Grim19.Use the Pubmed database to search for treatises related to STAT3 and Grim19.The protein associations and interactions between STAT3 and Grim19 was analyzed by GEPIA and String online tool.Download clinical cases in ovarian cancer from TCGA and study the subsistence analysis.By collating the clinical case data of the TCGA database,the online tool Linked Omics was used to screen related genes,and Omicshare was used to carry out GO function annotation enrichment analysis and KEGG signal pathway enrichment analysis of related genes.Results The expression levels of STAT3 and Grim19 are histologically specific.The expression of STAT3 was up-regulated in various malignant tumor diseases while the expression of Grim19 is down-regulated.The two protains are negatively correlated and specific interactive.TCGA data showed no significant difference between the median survival time of the STAT3 high expression group and the low expression group(P> 0.05).Grim19 high expression group had a longer median survival time than low expression group(P<0.05).2785 genes were related to STAT3(P <0.05),and the gene with the highest correlation coefficient was DNAAF2.4960 genes were related to Grim19(P <0.05),and the gene with the highest correlation coefficient was NDUFB7.GO show that STAT3 mainly involves biological processes as cellular processes,single-organism processes,metabolic processes,mainly involves molecular functions as binding,catalytic activity,signal transduction activity and mainly involves the cellular component as the cell part,cytoplasm and organelle.Grim19 mainly involves biological processes as cellular processes,metabolic processes,cell component organization or biogenesis,mainly involves molecular functions as binding,catalytic activity,structural molecular activity and mainly involves cellular component as cell part,organelle and organelle part.KEGG results show that the main cellular processes involved in related genes of STAT3 are decomposition and metabolism,the working organismal systems are the immune system and endocrine system,and the functions of the related genes of STAT3 are cell signal transduction functions and plays a significant role in many infectious system diseases and malignant tumors;The signal pathways of the related genes of Grim19 play an important role in neurodegenerative diseases,endocrine and malignant tumor diseases,and participate in various biological metabolic processes such as energy metabolism and nucleotide metabolism.Conclusion Grim19 may regulate the progress of ovarian cancer disease through STAT3;STAT3 plays an important role in the mechanism of tumor procession through multiple signaling pathways in ovarian cancer.Chapter 2.Expression and Clinical Significance of STAT3 and Grim19 in Epithelial Ovarian CancerObjective Explore the expressions of signal transducer and activator of transcription 3(STAT3)and gene associated with retinoid-interferon induced mortality(Grim19)in epithelial ovarian cancer(EOC),and the relationship with clinical pathological factors.Methods Ovarian tissue specimens resected through operation in Guangxi Medical University Afflicted Tumor Hosipital during 2013-2018 were collected,and the correlations of the expression levels of Grim19 and STAT3 with clinicopathological indexes were detected via immunohistochemistry(IHC)and Western blotting.Their positions in cells were observed through immunofluorescence.Results The IHC assay results showed that STAT3 had the lowest expression level in normal ovary(31.40 ± 1.25),followed by those in benign ovarian tumor(40.58±1.52)and borderline ovarian tumor(BOT)(52.07±3.02),but it had high expression in EOC(68.08 ± 1.20);The expression level of Grim19 was the lowest in EOC(14.17 ± 0.56),followed by those in BOT(19.47±1.12)and benign ovarian tumor successively(22.84±0.67),while it was highly expressed in normal ovary(37.55±1.02)(p<0.05);The expressions of the two genes presented negative correlations in all kinds of ovarian tissues(p<0.05).The expression level of STAT3 in EOC had no obvious correlations with FIGO staging,WHO classification(p>0.05).The expression level of Grim19 in EOC in stage FIGOIII-IV was higher than that in stage FIGOI-II;The expression levels of Grim19 in patients accepting NACT+IDS treatment in first visit were higher than those accepting PDS+ chemotherapy(p<0.05);Grim19 expression was not obviously associated with WHO classification(p>0.05).The expressions of Grim19 and STAT3 in lymphatic metastasis lesion had significantly positive correlations with the primary lesion(p<0.05).The Western blotting assay results were identical with the IHC assay results.The protein expression level of STAT3 and Grim19 had no obvious relationship with the overall survival of patients(P> 0.05).And the Cox model shows that age may be an independent risk factor affecting epithelial ovarian cancer(P <0.05).Immunofluorescence showed that STAT3 and Grim19 were mainly localized in the cytoplasm,and the two were co-localized on the mitochondria.Conclusion STAT3 presents high expression in EOC tissues while Grim19 is expressed in EOC tissues at low level.Grim19 may interact with STAT3 affected the biological process of ovarian cancer progression and metastasis;STAT3 and Grim19 may become potential therapeutic targets in overian cancer.Chapter 3.The effect of STAT3 and Grim19 on the function of ovarian cancer cell lines SKOV3 and A2780Objective To study the effects of p Grim19 combined with Si-RNA-STAT3co-expression plasmid(hereinafter referred to as p Grim19-si-STAT3)on the biological functions of ovarian cancer cell lines SKOV3 and A2780.Methods SKOV3 and A2780 cells were divided into four groups:experimental group(p Grim19-si-STAT3-SKOV3/A2780),blank plasmid control group(Empty plasmid control-SKOV3/A2780),blank liposome control group(Liposome control-SKOV3 /A2780),the parent strain control group(Control-SKOV3/A2780),and were transfected with the target plasmid,blank plasmid,and blank liposome,respectively.Western-blot was used to verify the transfection efficiency.CCK-8 experiment was used to verify the influence of cell proliferation ability.Wounding healing experiment was used to detected the influence of cell migeration ability.Results Western-blot showed that Grim19 was up-regulated in SKOV3 and A2780 experimental group while the expression of STAT3 and p STAT3 was down-regulated in SKOV3 and A2780 experimental group,indicating that the experimental group was successfully transfected with co-expression plasmid(P<0.05);CCK-8 results showed that after transfection with p Grim10-si-STAT3 plasmid,the cell proliferation ability of the SKOV3 and A2780 experimental group was inhibited(P<0.05);Wounding healing experiment results showed that high expression of Grim19 can decrease the migeration ability of overian cancer cells.Conclusion Overexpression of Grim19 may inhibit the proliferation and migration of ovarian cancer cell lines SKOV3 and A2780.