| ObjectiveHigh mortality of ovarian caner is primarily due to lack of early effectivescreening marker, and AAbs to TAAs have been shown to be present in the circulationof people with various forms of solid tumor very early in the disease process. Theimplications would be that AAbs to TAAs,which would provide a specificity andsensitivity blood test for early diagnosis of epithelial ovarian cancer. To increase thespecificity and sensitivity of CA125for the early detection and indeed to investigatethe further understanding in occurrence and development mechanism of ovarian cancer,we tested the efficiency of NFATc1auto-antibody alone and combination with CA125in epithelial ovarian cancer, and explored the expression of NFATc1antigens in thecancer, and the preliminary functions and mechanisms in SKOV3cells and nudemouse transplant tumor.Methods1All of the156patients were treated at the Department of Obstetrics andGynecology, First Affiliated Hospital, Chongqing Medical University between2010and2011. We diagnozed patients with ovarian tumors with different grades based onthe WHO and the International Federation of Gynecology and Obstetrics criteria.Preoperative serum samples from women with epithelial ovarian cancer and benigngynecologic disorders(include benign ovarian tumor and ovarian endometriosis cyst),and serum samples from healthy normal were obtained form Medical center of FirstAffiliated Hospital, Chongqing Medical University. These samples were stored at-80°C without any incident of thawing and the NFATc1antibody levels of differentgroups were tested by ELISA.2A total of156patient samples were collected from the First Affiliated Hospital of Chongqing Medical University, Department of Pathology, and were comprised ofthe following:78epithelial ovarian cancers,32benign ovarian tumors and46ovarianendometriotic cysts. Gynecologic pathologists at our institution made the histologicaldiagnoses. These formalin-fixed, paraffin-embedded tissues were sectioned at5μmthickness for standard immunohistochemical staining to detect the protein level ofNFATc1.SKOV3cell line (Human ovarian serous papillary adenocarcinoma) was gifedfrom The molecular medicine and tumor central, chongqing medical university.4-8weeks female nude mouse were provided from animal experiment central, chongqingmedical university. Gene and protein levels of NFATc1were detected from SKOV3cells and those transplant tumors before and after treated with NFATc1siRNA.Proliferation, apoptosis, invasion and migration of SKOV3cells (whichtreated/or without NFATc1siRNA) were tested by MTT,flow cytometer,Transwelland the erasion trace.Human ovarian cancer transplantation nude mouse were observed and theprogression of transplant tumors observed. The weight and the size of the transplanttumors have been compared before and after treated with NFATc1siRNA.3Blood and lymph vessels in transplant tumors before and after treated withNFATc1siRNA were signed by CD34and Podoplanin respectively,and then counted.Gene and protein levels of CXCR2, FGF-2and PDGF BB were detected in SKOV3cells and those transplant tumors before and after treated with NFATc1siRNA.Results1The mean of NFATc1autoantibody levels in normal controls, benign ovariantumors, ovarian endometriosis cysts and early stage of ovarian cancer patients,advanced stage of ovarian cancer patients were54.5658,67.4389,56.1318,145.5661and171.6052respectively. The difference between cancer cases and the other caseswas statistically significant (P＜0.05).On the basis of the cutoff value as P/N≥2.1, the number of positive cases innormal controls, benign ovarian cyst, ovarian endometriosis cyst and early stage of ovarian cancer patients, advanced stage of ovarian cancer patients were7,4,4,3,22,9respectively. The difference between cancer cases and the other cases was statisticallysignificant (P＜0.05). When the cutoff value as P/N≥2.1, early and advanced stages ofovarian cancer have a better diagnostic value, and the diagnosis of early stage ovariancancer seems to be more advantage.The diagnostic valve of the combination of serum NFATc1antibody and CA125were analyzed, and one of them is higher than the basis reference value is consideredpositive. The sensitivity in early stage of ovarian cancer was86.9%, the specificity was92.4%,and the diagnostic efficiency was88.2%, and the sensitivity in advanced stagesovarian was91.6%, the specificity was93.3%, and the diagnostic efficiency was88.2%, which higher than the detection of any one alone.2,The positive staining ratio of NFATc1was96%(75/78) in epithelial ovariancancer cases, and the HScore was269(0-356); whereas the positive ratio was18.75%(6/32) in ovarian benign cysts and the Hscore was75(0-180), and17%(8/46) inovarian endometriotic cysts and the Hscores were85, respectively(P﹤0.05). TheNFATc1was significantly higher in epithelial ovarian cancer than in ovarian benigndiseases (P﹤0.05).The expression of NFATc1RNA and protein in SKOV3cells and in transplanttumors were significantly dereference before and after treaded with NFATc1siRNA.The RNA OD value in cell control group is1.0846±0.0532; in siRNA group is0.5333±0.0208. The protein OD value in cell control group is0.9933±0.0577; insiRNA group is0.1896±0.0324.The proliferation of SKOV3cells were inhibited by NFATc1siRNA and the MTTshows that the inhibited ability was enhanced alone with the treated time and highest in48h.NFATc1siRNA promote SKOV3cells apoptosis and the value in control group is3.78±0.24%, in siRNA group is6.98±0.23%. The difference is significance (p=0.000).NFATc1siRNA reduce the invasion and migration of SKOV3cells. Transwellshows the cell number in control group(115±16；111±18) is significance than insiRNA group(39±7)(P <0.05). Scratch test shows the union speed of SKOV3cellsin siRNA group sooner than it in control groups in24and48hours (P <0.05).Inference of NFATc1siRNA in nude transplant epithelial ovarian tumor isstatistics significance. Transplant epithelial ovarian tumor grew in all18nude mouseand the weight and volume of tumor in siRNA group significant lower than which incontrol groups.3, The positive staining of NFATc1is express in cytoplasm of capillaryendothelial cells in epithelial ovarian cancer cell transplant tumor, and the values ofMVD and LMVD(12.00±1.65,11.47±0.32;10.03±0.96,9.95±1.12) in control groupswere significantly higher than which in siRNA group(5.36±0.34,4.67±0.26).The values of RNA and protein of NFATc1, CXCR2, FGF-2, and PDGF BB inSKOV3cells were significantly dereference before and after treaded with NFATc1siRNA. The RNA values of NFATc1, CXCR2, FGF-2, and PDGF BB in control groupswere0.912±0.001,0.925±0.005,0.895±0.005,0.913±0.005and0.896±0.001,0.882±0.003,0.893±0.001,0.873±0.001,1.0846±0.0532; and in siRNA group are0.265±0.003,0.338±0.001,0.296±0.001,0.218±0.003. The protein values in controlgroups are0.907±0.001,0.926±0.005,0.865±0.005,0.931±0.003；0.827±0.005,0.913±0.003,0.907±0.001,0.893±0.005; in siRNA group are0.295±0.003,0.382±0.001,0.396±0.001,0.318±0.003.ConclusionThe current study found that NFATc1auto-antibody and antigen werespecificially expressed in epithelial ovarian cancer patients. Combination of NFATc1antibody and CA125were the potential specific tumor markers for early epithelialovarian cancer. NFATc1antigen promoted the malignant biological behaves ofepithelial ovarian cancer and which probably rely on angiogenesis andlymphangiogenesis. In conclusion, our study would provide certain theory for earlydiagnosis, treatment,molecular mechanism and further research with epithelial ovariancancer. |
PDF Full Text Request