Molecular Mechanism Of WT1-mediated Epithelial Ovarian Cancer Metastasis

Ovarian cancer is a gynecological cancer having complex risk factors for its onset,metastasis and recurrence,with epithelial ovarian cancer,the main type of histopathology,accounting for 85%~90%.Although great efforts have been made in clinic to improve the effective treatment of epithelial ovarian cancer,currently the long-term effect is still not ideal.High recurrence and distant metastasis after treatment remain to be difficult problems to be solved in the field of obstetrics and gynecology.The Whilms tumor 1(WT1)can promote the tumorigenesis through various pathways,and plays an important role in tumor invasion and metastasis.Recent studies have shown that WT1 is highly expressed in epithelial ovarian cancer tissues and can be used as an important indicator for judging the degree of malignancy of tumors and assessing the poor prognosis.However,there are few reports on the molecular mechanism of WT1 regulating epithelial ovarian cancer invasion and metastasis.This study aimed to investigate the mechanism of WT1 in promoting epithelial ovarian cancer invasion and metastasis by means of databases,clinical specimens,in vitro cells and in vivo nude mouse models,and thus provide a new strategy for its clinical treatment.Part?Expression of WT1 in epithelial ovarian cancer and its correlation with clinical characteristicsObjective This section mainly studied the expression of WT1 in the clinical ovarian cancer chip database and cancer tissues of patients with epithelial ovarian cancer,and analyzes its relationship with the clinicopathological characteristics and prognosis of patients.Methods 1.Bioinformatics analysis: O ncomine was used to screen WT1 m RNA expression in ovarian cancer tissues and normal ovarian tissues through the classic tumor database.Simultaneously,Kaplan Meier Plotter database was used to analyze the relationship between WT1 and progression-free survival in ovarian cancer patients.2.Cancerous and adjacent tissues from 49 cases of epithelial ovarian cancer were collected and immunohistochemistry and q RT-PCR methods used to examine the expression of WT1 in epithelial ovarian cancer tissues and adjacent tissues.3.Statistical analysis was conducted of clinical data on the relationship between the m RNA expression level of WT1 and the pathological type,clinical FIGO stage,and pathological grade of patients with epithelial ovarian cancer.Additionally,we analyzed the correlation between WT1 and epithelial-mesenchymal transition molecular marker E-cadherin in epithelial ovarian cancer tissue.Results1.Analysis of oncomine ovarian cancer chip database showed that WT1 m RNA expression level was significantly up-regulated in ovarian cancer,and Kaplan Meier Plotter database analysis showed that patients with high expression of WT1 had significantly shorter progression-free survival compared with patients with low expression of WT1(P < 0.01).2.Immunohistochemistry and q RT-PCR showed that compared with adjacent tissues,the expression levels of WT1 protein and m RN A in cancer tissues of patients with epithelial ovarian cancer were significantly increased(P < 0.01).3.In patients with epithelial ovarian cancer,m RN A expression of WT1 in serous cystadenocarcinoma tissues was significantly higher than that of other subtypes(P < 0.05).The m RNA level of WT1 in advanced(stage III-IV)epithelial ovarian cancer tissues was significantly higher than that in the early stage(stage I-II);the m RNA expression level of WT1 increased as the pathological grade progressed(P < 0.05).4.Spearman rank correlation analysis showed that there was a negative correlation between the m RN A expression levels of WT1 and E-cadherin in epithelial ovarian cancer tissues(P < 0.01).Conclusion Based on chip database analysis of ovarian cancer and examination of clinical samples,WT1 is found to be significantly up-regulated in epithelial ovarian cancer,which is closely related to pathological type,clinical FIGO stage and pathological grade,and is the risk for progression-free survival,suggesting that WT1 may be involved in epithelial Malignant progress of ovarian cancer.Also,we believe that the m RNA expression levels of WT1 and E-cadherin in epithelial o varian cancer tissues are negatively correlated.It is speculated that WT1 may promote epithelial ovarian cancer invasion and metastasis through epithelial-mesenchymal transition.Therefore,further exploration of the functional mechanism of WT1 in epithelial ovarian cancer,especially the molecular mechanism of invasion and metastasis,is significant for clinical treatment,both theoretically and practically.Part II WT1 Promoting Invasion and Metastasis of Epithelial Ovarian Cancer Cells by Directly Targeting E-cadherinObjective The purpose of this part of the study was to verify the expression of WT1 in epithelial ovarian cancer cell lines.By investigating the effects and potential molecular mechanisms of WT1 on the migration and invasion of epithelia l ovarian cancer cells at the cellular level in vitro,we aimed to provide new targets and mechanisms for clinical treatment.Methods1.m RNA and protein expressions of WT1 in epithelial ovarian cancer cell lines SKOV3,HO8910,HO8910 PM,A2780,and normal ovarian epithelial cell line IOSE386 were detected by q RT-PCR and Western Blot methods,and cells with high and low expression of WT1 were selected before being subjected to subsequent tests.2.A WT1 knockdown stable cell line was constructed in the epithelial ovarian cancer cell line with high expression of WT1,and WT1 was overexpressed in cells with low expression of WT1.The effectivenessof knockdown and overexpression of WT1 was validated by q RT-PCR and Western Blot methods.3.The effects of WT1 knockdown and overexpression on the migration and invasion ability of epithelial ovarian cancer cells were observed using scratches and Transwell experiments.4.q RT-PCR and Western Blot was used to detect the effect of WT1 knockdown on the expression of the related molecular marks of epithelial-mesenchymal transition in epithelial ovarian cancer cells.5.The effect of WT1 on the promoter region of E-cadherin was investigated using Ch IP.6.Luciferase reporter gene experiments were done to verify the effect of WT1 on the activity of the target gene E-cadherin.Results1.Compared with the normal epithelial ovarian cell line IOSE386,q RT-PCR and Western Blot tests showed that the m RNA and protein expression levels of WT1 in epithelial ovarian cancer cell lines SKOV3 and HO8910 PM were significantly up-regulated(P <0.01),while the WT1 expression of A2780 was lower,and there was no significant difference compared with IOSE386(P > 0.05).2.q RT-PCR and Western Blot experiments confirmed that the expression of WT1 in SKOV3 and HO8910 PM knockdown cell lines was significantly down-regulated(P <0.01);while the protein expression of WT1 was significantly up-regulated in WT1 overexpressed A2780 cell lines(P <0.01).3.Cell scratches and Transwell experiments showed that down-regulation of WT1 significantly inhibited the migration and invasion of epithelial ovarian cancer cell lines,including SKOV3 and HO8910 PM cells.Conversely,overexpression of WT1 significantly promoted the migration and invasionof A2780 cells(P <0.01).Part III Mechanism of WT1 promoting invasion and metastasis of epithelial ovarian cancer cells via ERK1/2 signaling pathwayObjective This section aimed to explore the effect and mechanism of WT1 on signaling pathways in epithelial ovarian cancer cells.We hope to find existing molecular inhibitors for clinical treatment and improve the treatment and prognosis of epithelial ovarian cancer.Methods1.Western Blot was used to detect the p-AKT,AK T,p-ERK1/2 and ERK1/2 protein expression levels of SKOV3 cell lines with WT1 knockdown.2.p CMV-WT1 overexpressed plasmids,0ng,100 ng,500ng respectively,were transfected,and 1ugged in A2780 cells.Western Blot assay was used to detect the protein expression levels of p-ERK1/2,ERK1/2,and E-cadherin 48 h after transfection.3.Western Blot assay was used to detect the changes of p-ERK1/2,ERK1/2 and E-cadherin protein expression levels in different concentrations(0u M,10 u M,20 u M,30 u M)of ERK1/2 inhibitor(U0126)treated SKOV3 cells for 24 hours;in the mean time,the expression changes of p-ERK1/2,ERK1/2,and E-cadherin after U0126(20 u M)treated SKOV3 cells were detected at different time points(0h,6h,12 h,24h).4.After different concentrations(0u M,10 u M,20 u M)of U0126 were used to treat WT1 overexpressing A2780 cells,the effects of U0126 on the migration and invasion ability of WT1-induced epithelial-mesenchymal transition were detected using cell scratches and Transwell experiments.5.An in vivo assay was performed in xenograft mouse model of epithelial ovarian cancer: Nude mice were randomly divided into two groups,subcutaneously inoculated with SKOV3 cells and WT1 knockdown SKOV3 cells,and tumor volume was monitored weekly.After euthanizing the nude mice,the tumor tissues were retained and the knockdown of WT1 was verified by Western Blot.The liver tissues of the nude mice were photographed and the tumor metastasis was detected by HE staining.Western Blot was also used to detect the expression of p-ERK1/2,ERK1/2,E-cadherin and Vimentin in tumor tissues.Results 1.WT1 knockdown significantly inhibited ERK1/2 phosphorylation of SKOV3 cells(P < 0.01),and significantly up-regulated E-cadherin expression(P < 0.01),but had no significant effect on AKT phosphorylation(P > 0.05).2.WT1 overexpression significantly promoted the expression of p-ERK1/2 in A2780 cells,and inhibited the expression of E-cadherin in a concentration-dependent manner(P < 0.01).3.ERK1/2 inhibitor(U0126)reversed the decrease of E-cadherin expression level caused by WT1 time and dose-dependently(P < 0.01).4.U0126 inhibited the migration and invasion of epithelial ovarian cells induced by WT1,also concentration-dependently(P < 0.01).5.Western Blot verified the successful knockdown of WT1 in tumor tissues of nude mice,suggesting that down-regulation of WT1 effectively inhibited the liver metastasis of SKOV3 cells in nude mice.Western Blot analysis of tumor tissues also revealed that WT1 knockdown resulted in inhibition of p-ERK1/2 activation,while E-cadherin protein expression was significantly up-regulated.Conclusion WT1 can also activate the ERK1/2 signaling pathway of epithelial ovarian cancer cells,thereby inhibiting E-cadherin expression and participating in the migration and invasion of epithelial ovarian cancer cells.The in vivo assay performed in xenograft mouse model of epithelial ovarian cancer also demonstrates the role of WT1 ininvasion and metastasis of epithelial ovarian cancer.This section further elucidates the mechanism of WT1 in the invasion and metastasis of epithelial ovarian cancer,and provides new ideas of clinical diagnosis and treatment.In summary,the main findings of this study are: WT1 is highly expressed in epithelial ovarian cancer tissues and is closely related to the clinical FIGO stage,pathological type,pathological grade,and progression-free survival of patients.WT1 not only directly targets the promoter region of E-cadherin to regulate its transcription,but also inhibits the expression of E-cadherin by activating the ERK1/2 signaling pathway,thereby promoting the epithelial-mesenchymal transition process.O ur study elucidates the molecular mechanism of WT1 in promoting the invasion and metastasis of epithelial ovarian cancer cells,and provides new potential targets and strategies for clinical treatment of epithelial ovarian cancer.