| ObjectiveThe incidence of ovarian cancer in the female reproductive system malignant tumor ranks third,the mortality rate is the first,and the incidence is increasing year by year.Ovarian cancer cells have strong cell activity,rapid metastasis,lack of specific symptoms and signs,early warning of accurate molecular markers and difficulty in diagnosis.Claudins,a closely connected protein family,belongs to the membrane protein family,which is one of the important components of Tight junction(TJs).Claudins has abnormal expression in various diseases,and is closely related to the development of gynecological tumors such as cervical cancer,ovarian cancer and endometrial cancer.At present,claudin-6 is thought to be highly expressed in the pathologic tissue of ovarian cancer,indicating that claudin-6 may have different expression in ovarian cancer.By detecting claudins expression level and cellular space localization,it can be used as a practical indicator to diagnose and predict prognosis of gynecological malignant tumor.This article reviewed the relationship between claudins and gynecological tumors.This topic from cellular level to explore claudin-6 expression level in ovarian cancer,further reveal a express claudin-6 for ovarian cancer cell proliferation,migration apoptosis,etc.The influence of the biological characteristics,from the level of gene,protein validation claudin on outcome of ovarian cancer cell’s development,to further explore the role and mechanism of claudin-6 in ovarian cancer progression.Methods1.Fluorescence quantitative PCR and Western Blot were used to detect claudin-6 in SKOV3,A2780 and OVCAR3 ovarian cancer cell lines,as well as expression in the normal epithelial cell lines of IOSE80 ovary.2.Construct claudin-6 eukaryotic expression vector and transfection SKOV3 cells;3.Screening and identifying SKOV3 cells expressing claudin-6;4.CCK8,2D and 3D cloning experiment to detect the effect of claudin-6 overexpression on SKOV3 cell proliferation ability;5.Flow cytometry detected the effect of claudin-6 overexpression on SKOV3 cell apoptosis;6.Cell scratches and Transwell tests detected the effects of claudin-6 overexpression on the migration and invasion ability of SKOV3 cells;Results1.Claudin-6 is highly expressed in SKOV3,A2780 and OVCAR3 ovarian cancer cell lines,and expressed in the normal epithelial cell lines of IOSE80 ovary.2.PEGFP-N1-CLDN6 plasmid expressing EGFP fluorescent protein was constructed and identified.3.The claudin 6 transfection SKOV3 cell clone selection and identification of stable,by fluorescence microscope,real-time fluorescent quantitative PCR and Western blot method selected 3 stable express CLDN6 SKOV3 monoclonal,clone 1,7,and 10 respectively.Cell immunofluorescence staining showed that claudin-6 was expressed in cell membrane and nuclear membrane.4.CCK8 experiments showed that overexpression of SKOV3 cells proliferation rate than no-load group significantly enhanced,two-dimensional cloning formation experiment and three-dimensional form the experimental results showed that express group cell clone formation ability is empty cells significantly enhanced.5.The results of flow cytometry showed that the apoptosis rate of SKOV3 cells in the over-expression group was decreased and the time dependence was shown.6.The results of flow cytometry showed that claudin-6 had apoptotic inhibitory effect on SKOV3 cells of ovarian cancer.Conclusions1.Claudin-6 was highly expressed in ovarian cancer cell SKOV3 and OVCAR3,and the expression levels were low in ovarian cancer cell A2780 and normal ovarian epithelial cell IOSE80.2.Overexpression of claudin-6 promotes the proliferation of ovarian cancer cell SKOV3.3.Overexpression of claudin-6 enhanced migration and invasion ability of ovarian cancer cell SKOV3.4.Overexpression of claudin-6 has an apoptotic inhibition effect on ovarian cancer cell SKOV3. |
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