| Ovarian cancer is the most lethal gynecological malignancy,early symptoms are insidious and lack specificity,with a strong tendency of local infiltration and distant implantation and metastasis,more than 70%of patients are diagnosed at an advanced stage,with poor treatment.Metastasis and recurrence are leading causes of ovarian cancer treatment failure.However,the specific mechanism of high invasion and metastasis of ovarian cancer is not fully understood.Searching for molecular markers related to ovarian cancer metastasis and exploring its metastasis mechanism has always been a hot topic in ovarian cancer research.As a member of the Cancer-Testis Antigen(CTA)family,OY-TES-1 is specifically highly expressed in multisystem tumors,but not expressed in other normal tissues except testis.And has immunogenicity.Therefore,it is regarded as an ideal target for tumor therapy.However,the role and related mechanisms of OY-TES-1 in the occurrence and development of ovarian cancer remain unclear.Previous studies have found that OY-TES-1 could affect the malignant biological behavior of glioma and hepatoma cells.OY-TES-1 has KAZAL-1/2 domains that interact with transforming growth factor beta(TGF-β),and TGF-βmay be a downstream molecule of OY-TES-1.TGF-β is closely related to epithelial-mesenchymal transformation and tumor metastasis.Whether OY-TES-1 affects TGF-β and participates in the invasion and metastasis of ovarian cancer is still unclear.In addition,as a tumor associated antigen,OY-TES-1 can cause humoral immune response in some tumor patients,but its serum antibody is rarely studied in ovarian cancer.Therefore,from the following four parts,this study will preliminarily explore the expression and clinical significance of OY-TES-1 in ovarian cancer,its role in invasion and metastasis of ovarian cancer and its potential molecular mechanism,and explore the feasibility and clinical value of applying OY-TES-1 in auxiliary diagnosis and prognosis evaluation of ovarian cancer.In order to provide a scientific basis for the treatment strategy of intervening the invasion and metastasis of ovarian cancer and the selection of tumor markers for early diagnosis.Part Ⅰ Expression and clinical significance of OY-TES-1/TGF-β in ovarian cancerObjective To investigate the correlation between the expression of OY-TES-1 and TGF-β and the clinical parameters of ovarian cancer and the relationship with prognosis.Methods(1)The GEPIA and Oncomine databases were used to analyze the expression and correlation of OY-TES-1 and TGF-β in ovarian cancer and normal ovary.(2)The mRNA and protein expressions of OY-TES-1 and TGF-βin 65 ovarian cancer tissues and 20 normal ovarian tissues were detected by qRT-PCR and IHC,to analysis of the relationship between OY-TES-1 and TGF-β expression and clinical pathology,their correlation and the clinical significance of their co-expression.(3)The Kaplan-Meier method was used to analyze the relationship between the expression of OY-TES-1,the co-expression of OY-TES-1 and TGF-β and survival time of ovarian cancer patients,and plot the survival curve.Results(1)Database analysis showed that the expression of OY-TES-1 and TGF-β in ovarian cancer was significantly higher than that in normal ovary,and OY-TES-1 was significantly elevated in highly aggressive tumors and metastases.(2)The results of qRT-PCR showed that the expressions of OY-TES-1 and TGF-β were significantly up-regulated in ovarian cancer compared with normal ovarian tissues,and the two were positively correlated(r=0.462).IHC results showed that the positive expressions of OY-TES-1 and TGF-β were mainly located in the cytoplasm,and were strongly expressed in ovarian cancer tissues,while no OY-TES-1 was detected in the normal ovarian tissues.The high expression rates of OY-TES-1 and TGF-β in ovarian cancer were 70.7%and 69.2%,respectively.(3)In ovarian cancer,OY-TES-1 expression was positively correlated with FIGO stage and drug resistance;TGF-β expression was positively correlated with FIGO stage and lymph node metastasis(p<0.05).There was no significant correlation between the co-expression of the two and clinical parameters.(4)Survival analysis showed that patients with high OY-TES-1 expression and high co-expression of OY-TES-1 and TGF-β had shorter overall survival(OS)and disease-free survival(DFS)(p<0.05).Conclusion OY-TES-1 and TGF-β were highly expressed in ovarian cancer,and their expressions were positively correlated.High expression of OY-TES-1 and TGF-β is associated with FIGO stage,chemotherapy resistance and lymph node metastasis of ovarian cancer,and is associated with poor prognosis of patients.Both play an important role in the progression of ovarian cancer.OY-TES-1 may be a potential prognostic marker for ovarian cancer.Part Ⅱ The mechanism of OY-TES-1 regulating TGF-β expression in ovarian cancerObjective To analyze the effect of OY-TES-1 on TGF-β expression in ovarian cancer,to explore the mechanism of OY-TES-1 regulating TGF-βexpression.Methods(1)The changes of TGF-β after up/down-regulation of OY-TES-1 were verified by cell transfection and screening,qRT-PCR and Western blot.(2)Combined TargetScan,miRDB and miRanda database to screen target miRNAs shared both by OY-TES-1 and TGF-β,and predict the potential binding sites,qRT-PCR was used to detect the expression of target miRNA and analyze its correlation with OY-TES-1 and TGF-β.(3)The localization of OY-TES-1 and TGF-β with miRNA-326 in ovarian cancer cells was determined by fluorescence in situ hybridization(FISH).(4)Double luciferase reporter gene and RNA binding protein immunoprecipitation(RIP)assay were used to verify the target binding site and direct binding of OY-TES-1 and TGF-β to miRNA-326.(5)Rescue experiment,qRT-PCR,Western blot and Double luciferase reporter gene assay were used to verify that the regulation of TGF-β expression by OY-TES-1 was dependent on miRNA-326.Results(1)qRT-PCR and Western blot showed that OY-TES-1 positively regulated TGF-β2 expression.(2)The database screened out that OY-TES-1 and TGF-β shared miRNA-326,and both of them have specific binding sites of miRNA-326 in the 3’UTR region.The detection showed that the expression of miRNA-326 decreased in ovarian cancer tissue and was negatively correlated with the expression of OY-TES-1 and TGF-β(r=-0.274,r=-0.52).(3)FISH experiments showed that OY-TES-1 and TGF-β co-localized with miRNA-326 in the cytoplasm.(4)Double luciferase reporter gene verified the targeted binding between OY-TES-1 and miRNA-326,TGF-β and miRNA-326.RIP assay confirmed that the direct binding of OY-TES-1 and TGF-β to miRNA-326 involved the involvement of RNA-induced silencing complex(RISC).(5)miRNA-326 can negatively regulate the expression of OY-TES-1 and TGF-β.The regulation of TGF-β expression by OY-TES-1 depended on miRNA-326,and OY-TES-1 3’UTR affected the activity of TGF-β 3’UTR through miRNA-326,thus regulating the expression of TGF-β.Conclusion In ovarian cancer cells,OY-TES-1 can act as ceRNA and regulate TGF-β expression by competitively binding miRNA-326.Part Ⅲ Effects of OY-TES-1/miRNA-326/TGF-β axis on ovarian cancer invasion and metastasis by mediating EMT and angiogenesisObjective To analyze the effect of OY-TES-1/miRNA-326/TGF-βregulatory axis on EMT and angiogenesis,and to explore its role in ovarian cancer invasion and metastasis.Methods(1)The ovarian cancer cell lines with stable up-regulation and down-regulation of OY-TES-1 were constructed by lentiviral transfection,screened and verified the transfection efficiency.(2)The effect of OY-TES-1 expression level on cell morphology observed by microscopy.(3)The effects of OY-TES-1 and miRNA-326 on migration,invasion and angiogenesis of ovarian cancer cells were verified by wound healing assay,transwell migration assay,matrigel invasion assay and angiogenesis assay.The effects of miRNA-326 and TGF-β on OY-TES-1 action were determined by rescue experiment.(4)The effects of OY-TES-1 on the tumorigenesis and peritoneal metastasis of ovarian cancer cells in vivo were evaluated by constructing subcutaneous and peritoneal tumorigenesis animal models in nude mice.And qRT-PCR,Western blot and IHC were used to detect the expression of OY-TES-1,TGF-β,EMT and angiogenesis-related indexes in tumor tissues.Results(1)Successfully constructed shRNA-OY-TES-1 SKOV3,A2780 and Lv-OY-TES-1 HO8910 stable transfection cell lines,the expression of OY-TES-1 in the down-regulated group was significantly lower than that in the negative control group,and the expression of OY-TES-1 in the up-regulated group was significantly higher than that in the negative control group(p<0.05).(2)Down-regulation of OY-TES-1 made the cell morphology tend to be epithelial.Up-regulation of OY-TES-1 made cell morphology tend to be mesenchymal cells.(3)In vitro tests showed that down-regulation of OY-TES-1 inhibited the ability of ovarian cancer cells to migrate,invade and angiogenesis,which was accompanied by increased expression of E-cadherin and decreased expression of N-cadherin,Vimentin,VEGF and Angiopoietin-1,and up-regulation of OY-TES-1 does the opposite.Overexpression of miRNA-326 inhibits the migration,invasion and EMT process of ovarian cancer cells.Inhibition of miRNA-326 or overexpression of TGF-β can reverse the inhibitory effect of low OY-TES-1 expression on ovarian cancer cell migration and invasion,and overexpression of TGF-β can also reverse the inhibitory effect of low expression of OY-TES-1 on angiogenesis.Overexpression of miRNA-326 or inhibition of TGF-β can reverse the promotion of OY-TES-1 overexpression on ovarian cancer cell migration and invasion,and inhibition of TGF-β also reversed the promoting effect of OY-TES-1 overexpression on angiogenesis.The expression changes of EMT-related signature molecules caused by up-/down-regulation of OY-TES-1 could be partially reversed by miRNA-326 mimics/inhibitors.(4)In vivo experiments showed that down-regulation of OY-TES-1 inhibited tumorigenicity and peritoneal implantation metastasis of ovarian cancer cells in vivo,and inhibited ascites formation.After down-regulating the expression of OY-TES-1 in ovarian cancer cells,the expression of E-cadherin in tumorigenic tissues increased,while the expressions of N-cadherin,Vimentin,VEGF and Angiopoietin-1 decreased.Conclusion OY-TES-1 promotes the migration,invasion and angiogenesis of ovarian cancer cells and promotes the EMT process.miRNA-326 inhibits the migration and invasion of ovarian cancer cells and inhibits the EMT process.The OY-TES-1/miRNA-326/TGF-β axis can induce EMT and angiogenesis to promote the invasion and metastasis of ovarian cancer cells.Part Ⅳ Clinical significance analysis of serum OY-TES-1 antibody in ovarian cancer patientsObjective To investigate the presence of OY-TES-1 antibody in serum of ovarian cancer patients,to explore the clinical significance and application value of OY-TES-1 serum antibody.Method(1)ELISA was used to detect OY-TES-1 antibody and protein in sera of 56 ovarian cancer patients,and 40 healthy controls.(2)The receiver operating characteristic curve(ROC curve)was drawn to evaluate the value of serum OY-TES-1 antibody in the diagnosis of ovarian cancer,and the diagnostic performance of the combined detection of serum OY-TES-1 antibody and CA125.(3)The correlation between OY-TES-1 antibody and clinical parameters and patient prognosis was also analyzed.Results(1)ELISA showed that the positive rates of serum OY-TES-1 antibody and protein in ovarian cancer patients were 28.5%and 7.1%,respectively.Normal human serum is negative.(2)The area under the ROC curve of serum OY-TES-1 antibody for the diagnosis of ovarian cancer was 0.802(95%CI=0.708-0.876,p<0.001),the sensitivity was 85.71%,and the specificity was 55.0%.The area under the ROC curve of serum CA125 for the diagnosis of ovarian cancer was 0.900(95%CI=0.822-0.952,p<0.001),the sensitivity was 99%,and the specificity was 80%.The area under the ROC curve of serum OY-TES-1 antibody combined with CA125 in the diagnosis of ovarian cancer was 0.960(95%CI=0.899-0.990,p<0.001),the sensitivity was 96.43%,and the specificity was 90%.(3)There was no significant correlation between the presence of OY-TES-1 antibodies and clinical parameters.Survival analysis showed that there was no significant correlation between the presence of OY-TES-1 antibody and patients’ OS and DFS.Further analysis of OY-TES-1 protein expression in tumor tissues and OY-TES-1 antibody in serum showed no significant correlation with patients’ OS.Conclusion The specific antibody of OY-TES-1 appeared in the serum of some patients with ovarian cancer,with high diagnostic performance and sensitivity.The serum OY-TES-1 antibody has certain clinical value in the screening and diagnosis of ovarian cancer,it combined detection with CA125 can improve the diagnostic efficiency of ovarian cancer,and this may become a specific tumor marker combination for early diagnosis of ovarian cancer. |
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