| Epithelial Ovarian Cancer(EOC)is the leading cause of gynecological cancer related death.Most radical resection of EOC can get with the development of science and technology and surgical techniques,but the overall survival rate still remains low.The most important factor is that EOC extremely had the ability of invasion and metastasis.Once metastasis happens,the survival rate of EOC will drop sharply.Therefore,study on the mechanism of EOC invasion and metastasis has always been the scientific hotspots.Epithelial-mesenchymal transition(EMT)refers to the biological process that epithelial cells transfer into mesenchymal phenotype cells and have their characteristics.Currently,most scholars regarded that EMT is an important way of tumor invasion and metastasis.Chemokine CXCL12,which was also called stromal cell-derived factor-1,belongs to chemokine CXC family and combines with their receptors to promote tumor cell's growth,proliferation and invasion or directly recruits endothelial progenitor cells to tumor angiogenesis.Some papers already proved that chemokine CXCL12 could play a critical role in EOC progression and metastasis.Anoikis is a special type of programmed cell death(PCD)which could be induced by the detachment of cell-extracellular matrix or cell-cell.Several studies show that anoikis takes role in the process of epithelial cells malignant transformation in many tissues.Anoikis resistance could increase the survival time of tumor cells after its detachment from primary lesion which is regarded as the first step to tumor metastasis.Based on the above background,we hypothesized whether chemokine CXCL12 could promote EOC cells invasion and metastasis in an EMT way and,it the meantime,increase the anoikis resistance of EOC cells.In order to verify our hypothesis,the study mainly included in the following aspects:(1)Using Westren blot,We tested the expression of CXCR4 and CXCR7 in three ovarian cancer cell lines(SKOV3,HO-8910,A2780).(2)Through MTT,Transwell and wound-healing assays,we tested the change of proliferation,invasion and migration of EOC cancer cells after its treatment with CXCL12.(3)We used optical microscope to detect the morphological changes of EOC cells after its co-culture with CXCL12.(4)By flow cytometry,we tested EOC cells apoptosis rate before and after the treatment with CXCL12(5)We detected the expression difference of EMT-associated protein and apoptosis-associated protein by Western blot,and then concluded that CXCL12 played role mainly through the combination with CXCR7.(6)By immunohistochemistry,CXCR7 protein were detected in normal ovarian tissues,benign ovarian tumor tissues and malignant ovarian tumor tissues.(7)At mean time,clinicopathological data and follow-up data of EOC patients were also collected.In order to obtain the correlation between CXCR7 and clinical outcomes,data analysis and statistical analysis were performed.The study is divided into three parts.Part ? CXCL12 could promote the ability of proliferation,invasion and metastasis in EOC cells.Objective To clarify CCL20 can promote the proliferation,invasion and metastasis in EOC cells.Methods First,all the three ovarian cancer cell lines(SKOV3,HO-8910,A2780)expressed CXCR4 and CXCR7.Next,by MTT,we detected the changes of proliferation index in vitro after CXCL12 co-cultured with different ovarian cancer cell lines(SKOV3,HO-8910 and A2780)for 24 h;Last,through Transwell and wound-healing test,we tesed the invasion and migration changes in EOC cells.Results MTT revealed that CXCL12 could improve the proliferation rate of EOC cells in a concentration-dependent manner.Transwell also showed that CXCL12 could promote the migration of the EOC cells in a concentration-dependent manner.Furthermore,Wound-healing test verified that CXCL12 could also increase the migration ability of EOC cells.Conclusions CCL20 could promote EOC cells' proliferation,invasion and metastasis.Part ? The mechanism on CXCL12-induced ovarian cancer cell biological changesObjective To clarify the mechanism on CXCL12-induced ovarian cancer cell biological changesMethods First,we used optical microscope to observe tumor cell morphologic changes aftre CXCL12 co-cultured with ovarian cells(SKOV3,HO8910 and A2780)for 48h;Next,the changes of tumor cell survival rate and quantity were observed after treatment with CXCL12 for 48 h by flow cytometry test;Then,through Western blot,we tesed the expression changes of EMT-related and anoikis-related proteins;Finally,the apoptosis effect of CXCL12 on EOC cells was tested by flow cytometry.Results Through optical microscope,with 100 ng/m L CXCL12 treatment for 48 h,EMT changes were induced by SKOV3 and HO-8910,but not by A2780 apparently;In anoikis condition,we added 100mg/m L CXCL12 for 48 h and then found increased spheriods number,decreased apoptosis rate;Through Western blot,we could see that the expression of CXCR4 and CXCR7 were all up-regulated and interestingly,compared to CXCR4,CXCR7 was more obviously up-regulated.In the meantime,we also found that the up-regulated expression of Vimentim,?-catenin and Bcl-2 while the expression of E-cadherin,caspase3 and Bax were down-regulated;Using flow cytometry,we could find that there are significant decrease in the sum of the second quadrant and the fourth quadrant after the treatment of CXCL12 for 48 h.Conclusions CXCL12 could promote the metastasis and survival rate in an EMT and anoikis resistance maner through the combination mainly with CXCR7 and/or CXCR4.Part ? Clinical significance of CXCR7 expression and its correlation with prognosis of ovarian carcinomaObjective To detect the expression of CXCR7 in EOC tissues and determine the relationship between the clinicopathological data and prognosis of EOC pattientsMethods Using Immunohistochemistry test to identify the expression of CXCR7 in different ovarian tissues(normal ovarian tissues,benign ovarian tumor tissues and malignant ovarian tumor tissues)and analyzed their date with CXCR7 expression and clinicopathological data.Using univariate and multivariate analysis to predict poor prognosis markers of EOC patient.Using Kaplan-Meier method and log-rank test to compare the overall survival rate and disease-free survival rate in the two groups.Results CXCR7 expression mainly loated in EOC cell membrane and cytoplasm and about 68.42%(39/57)CXCR7 expressed in EOC tissues,but only 26.7%(4/15)and 13.3%(2/15)CXCR7 expressed in benign ovarian tumor tissues and normal ovarian tissues,respectively.Comparing with other two groups,the expression of CXCR7 in EOC tissues showed significant statistical significance.We also found that CXCR7 expression was significantly correlated with the FIGO stage,histological grade,lymph node metastasis and peritoneal metastasis(p<0.05);and age,preoperative CA125 level,pathological type of ovarian cancer,residual tumor diameter,ascites content,tumor size and location had no significant relationship(p>0.05).Using univariate and multivariate Cox regression analysis,we could find that CXCR7 expression,FIGO stage,histological grade,lymph node metastasis and peritoneal metastasis can significantly affect overall survival and disease-free survival in EOC patients.Comparing low expression group,high CXCR7 expression group showed significantly decreased overall survival(P=0.025)and disease-free survival(P=0.043).Conclusions CXCR7 over-expressed in EOC and high expressed CXCR7 correlated with poor prognosis in EOC patients which suggested that CXCR7 could possibly predict the clinical outcomes of EOC patients and might be a new target in the treatment of EOC.Summary CXCL12 can enhance the ability of proliferation,invasion and metastasis,and anoikis resistance of EOC cells by inducing tumor cells into EMT-like changes and inhibition of anokisis mainly through CXCR7.Clinical test demonstrated that high CXCL12 expression in EOC tissues was correlatted with clinicopathologic data and the overall survival and disease-free survival.Therefore,we can conclude that CCL20 may become a potential prognostic indicator to guide individual treatment of EOC patient. |
PDF Full Text Request