The Study On The Relationship Between TFF1 Of Epithelial Ovarian Cancer Cells And Chemoresistance Replacemented Drugs Research Effects On Epithelial Ovarian Cancer Cells

Ovarian cancer is a serious global threat to female health and is a leading cause of cancer-related mortality in females,often due to late-stage recognition and aggressive tumor relapse.Epithelial ovarian cancer（EOC）arises from the ovarian surface epithelium（OSE）and account for greater than 90% of all ovarian cancers.EOC is the leading cause of gynecological cancer mortality.Among the gynecological cancers,ovarian cancer is less common than cancer of the cervix and the uterus,but it has the highest fatality-to-case ratio,accounting for almost half of the deaths from cancers of the female genital tract.The standard treatment is satisfactory cytoreductive surgery and platinum-based combination chemotherapy,with taxol and carboplatin being the first line choice.In spite of the standard therapy the prognosis of EOC patients is not favorable and the 5-year survival rate for advanced patients（stage III and IV）is only 30%.Compared with the progress in surgery and chemotherapy,the progress in understanding the pathogenesis of EOC grows very slowly.Chemoresistance has emerged as the major obstacle of failure in the treatment of ovarian cancer patients.The complete response rate of chemotherapy is less than 75% and 15-30%.EOC patients does not response to the fist line regime.Among the cases displaying OR more than 90% patients will suffer from recurrence.It is vital to predictive chemoresistance and improve the outcome of therapy.The current criterion of chemoresistance is based on the clinical chemotherapy outcome of chemotherapy and cannot be detected before the determination of chemotherapy regime.Therefore,it is urgent to explore an effective predictive marker of platinum-resistance to guide the application of platinum before the treatment or at the early stage of chemotherapy.Early on,studies started to focus on genetic and molecular mechanisms that mediate resistance.Recently,objective to discuss the role of sensitizer of chemotherapeutic drugs,especially,the mechanism of platinum resistance is being explored and novel targets are being identified.On the basis of ovarian epithelial tumors of the WHO classification,EOC could be classified: serous tumors,mucinors tumors,endometrioid tumors,clear cell tumor of ovary,Brenner tumor and so on.The most common pathological type is the serous tumors.They are different tissue types,and different biological characters.The trefoil factor family（TFF）is a relatively new family of peptides which bear the three-loop trefoil domain.To investigate the effect of stable up-regulation of trefoil factor 1（TFF1）gene on the migration and invasion ability of epithelial ovarian cancer.Trefoil factor（TFF）family is a group of small peptides that has one or more trefoil structural domain,which is synthesized and secreted mainly by the digestive tract mucous secretory epithelial cells and plays a major role in the process of mucosa damage repair.In recent years,the correlation between TFF family and tumorigenesis,invasion and clinical prognosis has widely been drawing attention.There were more studies on correlation between TFF1 expression and oncogenesis,progression and prognosis of breast and stomach cancer.But reports on relationship between TFF1 expression and EOC occurrence,development and clinical prognosis are rare.1α,25-Dihydroxyvitamin D3（1,25（OH）₂D₃）has been demonstrated to inhibit the growth of cancer cells.It is the most active metabolite of vitamin D3.More recent findings suggest that 1,25（OH）₂D₃ is an important modulator of cellular proliferation and differentiation in a variety of benign and malignant cells.It is also exhibits anti invasion,anti-angiogenesis,and anti-metastatic activity in vivo.Some findings suggested that 1,25（OH）₂D₃ suppresses SKOV3 growth and enhances the anti-proliferative effect of carboplatin,and the drugs function synergistically by inducing cell cycle arrest,suppressing proliferation.Doxorubicin is the most important chemotherapeutic in clinical therapy.There is a new type chemotherapeutic called liposome doxorubicin,was used to be the second-line drugs to treatment the ovarian cancer.But reports on relationship between TFF1 expression and EOC occurrence,development and clinical prognosis are rare.And there is few report about Synergy of 1,25-dihydroxyvitamin D3 and liposome doxorubicin in growth suppression of SKOV3 cells.The research contents and results are as follows:Part I The study on the chemoresistance between mucinous ovarian cancer cells and the expression of Trefoil factor 1.Objective:To understand expression of TFF1 in mucinous ovarian cancer cell and its relations with chemoresistance.Methods: We used different mucinous ovarian cancer cell lines.To transfection the cells with RNA isolation and quantitative real-time PCR,to observe the expression of TFF1 in mucinous ovarian cancer cell and its relations with chemoresistance.Results:1 Examined the m RNA and protein level of TFF1 in mucinous ovarian cancer cell lines.We found that both the m RNA and protein level of TFF1 were highly expressed in OMC-3 and MCAS cells.2 Silenced TFF1 in OMC-3 and MCAS cells could knockdown the level of TFF1 in Caov-3 cell.3 TFF1 promotes mucinous ovarian cancer cell chemoresistance to cisplatin.Considering that chemoresistance is a malignant characteristic in ovarian cancer,we then used these transfected cell lines performed chemosensitivity assays.We treated OMC-3 cells with different concentration（4ug/m L,6ug/m L）of cisplatin and then examined the cell viability and we found that TFF1 reduces ovarian cancer cells the ability of chemoresistance to cisplatin（P<0.01）.We treated MACS cells with different concentration（2ug/ m L、4ug/ m L、6ug/ m L）of cisplatin and then examined the cell viability and we found that TFF1 reduces ovarian cancer cells the ability of chemoresistance to cisplatin（P<0.01）.4 Performed CCK-8 cell viability assays.TFF1 elevates the abilities of cell viability and invasion in mucous ovarian cancer.We Surprisingly,loss function assays revealed that TFF1 significantly elevates the cell viability.We then used these transfected cell lines performed transwell cell invasion assays,and we found that TFF1 also remarkably promotes ovarian cancer cell invasion.These findings remind us that TFF1 plays an aggressive role in mucinous ovarian cancer progression.Conclusion:1 Both m RNA and protein level of TFF1 is high expression in mucinous ovarian cancer cell line OMC-3 and MCAS.2 TFF1 elevates mucous ovarian cancer cells the ability of chemoresistance..3 TFF1 promotes mucous ovarian cancer cells invasion.Part II The study on the chemoresistance between serous ovarian cancer cells and the expression of Trefoil factor 1.Objective: To understand expression of TFF1 in serous ovarian cancer cell and its relations with chemoresistance.Methods:We used different serous ovarian cancer cell lines.To transfection the cells with RNA isolation and quantitative real-time PCR,to observe the expression of TFF1 in serous ovarian cancer cells and its relations with chemoresistance.Results:1 We examined the m RNA and protein level of TFF1 in serous ovarian cancer cell lines.We found that both the m RNA and protein level of TFF1 were lowly expressed in Caov-3 and SKOV3 cells.2 We examined the m RNA and protein level by overexpression of TFF1.We founded the TFF1 were remarkably altered the level of TFF1 in Caov-3 cell.3 TFF1 promotes serous ovarian cancer cell chemoresistance to cisplatin.Considering that chemoresistance is a malignant characteristic in ovarian cancer,we then used these transfected cell lines performed chemosensitivity assays.We treated Caov-3 cells with different concentration（2ug/ m L、4ug/m L、6ug/ m L and 8ug/ m L）of cisplatin for 1.5 hours and then examined the cell viability and we found that TFF1 enhances ovarian cancer cells the ability of chemoresistance to cisplatin（P<0.01）.4 Performed CCK-8 cell viability assays.TFF1 elevates the abilities of cell viability and invasion in serous ovarian cancer.We Surprisingly,gain function assays revealed that TFF1 significantly elevates the cell viability.We then used these transfected cell lines performed transwell cell invasion assays,and we found that TFF1 also remarkably promotes ovarian cancer cell invasion.These findings remind us that TFF1 plays an aggressive role in serous ovarian cancer progression.Conclusion:1 Both m RNA and protein level of TFF1 is low expression in serous ovarian cancer cell line Caov-3 and SKOV3.2 Elevates the level of TFF1 promotes Caov-3 cell viability in indicated concentration of cisplatin.3 TFF1 promotes serous ovarian cancer cell invasion.Part Ⅲ The study on the relationship between TFF1 of serous ovarian cancer cells and replacemented drugs research effects on serous ovarian cancer cells.Objective: To understand the the relationship between TFF1 of serous ovarian cancer cells and replacemented drugs research effects on serous ovarian cancer cells.Methods: In these experiments,cell viability was determined by MTT,and the espress of protein by western blot.The aim to examine the survival and the apoptosis rate of SKOV3 cells.To transfection the cells with RNA isolation and quantitative real-time PCR,to observe the expression of TFF1 in serous ovarian cancer cells.Results:1 In the cell viability assay,1,25（OH）₂D₃ inhibited growth in a dosedependent manner.Combined administration of 1,25（OH）₂D₃ and liposome doxorubicin,the growth inhibition was significantly greater with the combined treatment.1,25（OH）₂D₃ and liposome doxorubicin inhibited growth in a dose-dependent manner.2 Apoptosis was assessed to identify the mechanism of growth inhibition by the combined treatment of 1,25（OH）₂D₃ and liposome doxorubicin in ovarian cancer cells,1,25（OH）₂D₃ and liposome doxorubicin inhibited growth in a dose-dependent manner.3 We found that the express of Caspase-3 is up-regulated in epithelial ovarian cancer.And the level of PNCA is down-regulated express.4 We examined the m RNA level of TFF1 in SKOV3 cells.We found that the m RNA level of TFF1 were lower expressed in combined drugs group than 1,25（OH）₂D₃ group and liposome doxorubicin group.Conclusion:1 1,25（OH）₂D₃ and liposome doxorubicin both have obvious inhibitory effect on ovarian cancer cell lines of SKOV3,and its inhibition effect has a dosage and time dependent.2 1,25（OH）₂D₃ and liposome doxorubicin can increase SKOV3 cells apoptosis.3 Caspase-3 and PCNA could make a important role in SKOV3 cells apoptosis.4 TFF1 could promotes the inhibition effect of that 1,25（OH）₂D₃ suppresses SKOV3 growth and enhances the anti-proliferative effect of liposome doxorubicin.