To Study The Role And Mechanism Of CT-OATP1B3 On Human Ovarian Cancer Cells

Objective: In this study,the expression and localization of Ct-OATP1B3(Cancer-type Organic Anion Transporting Polypeptide 1B3)in epithelial ovarian cancer cells were first detected,and analyze the relationship between Ct-OATP1B3 and ovarian cancer development and prognosis.Second,the biological function of Ct-OATP1B3 was observed in epithelial ovarian cancer cell line and the molecular mechanisms was discussed.Methods:1.Immunohistochemical method was used to detect the expression of Ct-OATP1B3 in different clinical staging of epithelial ovarian cancer organization.Combined with TCGA database,the correlation between the expression of Ct-OATP1B3 and the development and prognosis of ovarian cancer was analyzed.2.The human epithelial ovarian cancer cell line SKOV3 and OVCAR3 were selected and the the human normal ovarian epithelial cells HOSE was for control.The expression and localization of Ct-OATP1B3 in ovarian cells were observed by RT-PCR and cellular immunofluorescence.3.On the basis of epithelial ovarian cancer cell line SKOV3 and OVCAR3,Ct-OATP1B3 overexpression and knockdown cell model were constructed.Use the CCK-8,flow cytometry,cell scratch experiment,cell adhesion and Transwell chamber invasion experiment to analysis of Ct-OATP1B3 impact on the biological functions of ovarian cancer cells.4.In normal controls,Ct-OATP1B3 overexpression and knockdown SKOV3 and OVCAR3 cell lines,use RT-PCR and Western blot methods to analysis the effects of Ct-OATP1B3 on expression of the epithelial markers E-Cadherin and Keratin 18,and expression of the mesenchymal markers Fibronectin and FSP1.The molecular mechanism of Ct-OATP1B3 function was preliminarily discussed from the perspective of Epithelial and Mesenchymal Transition(EMT).Results:1.This study included 87 cases of human normal ovarian epithelial tissue or epithelial ovarian carcinoma.Immunohistochemical staining results showed thatImmunohistochemical staining results showed that the positive rate of Ct-OATP1B3 in normal ovarian tissue was 0%.According to the American cancer society TNM staging,in phase ?,phase ?,phase ? ovarian cancer tissues,Ct-OATP1B3 expression level significantly increases,the positive rate of expression was 25.0%,57.9% and 83.3%(P<0.05),respectively.The average expression of Ct-OATP1B3 in ovarian cancer tissues was significantly higher than that of normal ovarian tissue.The highest expression level in phase ? ovarian cancer tissue,significantly higher than ? phase(P <0.01)and ? period(P <0.05).Compare with ? phase,expression of Ct-OATP1B3 in ? phase was only increasing,but not significant difference(P=0.8649).Among 54 patients with ovarian cancer who received follow-up data,the overall survival rate of patients with expression positive of Ct-OATP1B3 was significantly lower than patients with expression negative of Ct-OATP1B3(P<0.001).The results of TCGA database also showed that Ct-OATP1B3 expression increased with the increase of clinical stage of ovarian cancer patients.And the overall survival rate of patients with expression positive of Ct-OATP1B3 was significantly lower than patients with expression negative of Ct-OATP1B3.2.Real-time PCR showed that,Ct-OATP1B3 mRNA expressed in normal ovarian epithelial cells was extremely low.Compared with normal ovary cell HOSE,the mRNA levels of Ct-OATP1B3 in ovarian cancer cells OVCAR3 and SKOV3 cells were significantly higher,which were 687.9 and 591.7 times of the mRNA levels in the HOSE cells respectively(P<0.01).Cellular immunofluorescence results showed that Ct-OATP1B3 was mainly expressed in the cytoplasm of ovarian cancer cells OCVCR3 and SKOV3.3.Experimental of cell biological behavior found that compared with the control cells SKOV3 and OVCAR3,after overexpression of Ct-OATP1B3,the growth and proliferation capacity of cells were increased by 1.134 and 1.114 times respectively(all P<0.05),the migration rate of cells were increased by 2.03 times and 1.65 times respectively(all P<0.01),the cell invasion rate were increased by 1.75 times and 2.27 times respectively(all P<0.01).After knockdown of Ct-OATP1B3,the proliferation ability of cells were reduced by 9.03% and 9.34% respectively(P<0.05),the migration rate of cells were reduced by 51.3% and 58.0% respectively(P<0.01),the cell invasion rate were reduced by 38.4% and 41.7% respectively(P<0.01).Ct-OATP1B3 had no significant effect on apoptosis and adhesion ability of ovarian cancer cells.4.Compared with control group of SKOV3 and OVCAR3 cells,after overexpression of Ct-OATP1B3 in SKOV3 and OVCAR3 cells,showed that the EMT epithelial cells markers E-Cadherin mRNA levels reduced by 70.3% and 77.9%(all P<0.001),epithelial cells markers Keratin 18 mRNA levels reduced by 29.7% and 36.4% respectively(P<0.01,P<0.05)mesenchymal cells markers Fibronectin mRNA level increased 4.38 times and 6.13 times(all P<0.001),mesenchymal cells markers FSP1 mRNA level increased 2.07 times(P<0.05)and 1.19 times,respectively.And Compared with control group,after knockdown of Ct-OATP1B3 in SKOV3 and OVCAR3 cells,showed that the EMT epithelial cells markers E-Cadherin mRNA levels increased 1.68 times and 1.94 times(P<0.05,P<0.01),epithelial markers Keratin 18 mRNA level increased 1.16 times and 1.25 times respectively,mesenchymal cells markers Fibronectin mRNA levels reduced by 60.7% and 58.7%(P<0.001),mesenchymal cells markers FSP1 mRNA levels reduced by 10% and 16% respectively,(figure 2.9 A,B).Western blot experiment further verified that,compared with control group,after overexpression of Ct-OATP1B3 in SKOV3 and OVCAR3 cells,found that the EMT epithelial cells markers E-Cadherin protein levels reduced by 23.2% and 34.5%(all P<0.05),respectively,Fibronectin mesenchymal markers protein levels increased 1.51 times and 1.92 times(P<0.05,P<0.01),respectively.And Compared with control group,after knockdown of Ct-OATP1B3 in SKOV3 and OVCAR3 cells,showed that the EMT epithelial cells markers E-Cadherin protein levels increased 1.69 times and 1.25 times(P<0.01,P<0.001),respectively,and the level of Fibronectin mesenchymal markers protein was reduced by 15.4% and 31.2%(all P<0.01),respectively.Conclusion: Compared with the normal epithelial ovarian tissue cells,the expression of Ct-OATP1B3 was sifnificantly up-regulated,main located in the cytoplasm.It was positively correlated with the clinical staging of ovarian cancer patients and was negatively correlated with the overall survival rate of patients.Ct-OATP1B3 mainly promotes the migration and invasion ability of epithelial ovarian cancer cells,and also has a weak enhancement effect on cell growth and proliferation ability.Ct-OATP1B3 may play an important role in the migration and invasion of ovarian cancer cells by promoting the EMT process of ovarian cancer cells.