GOLPH3 Induces Epithelial-mesenchymal Transitionvia The Wnt/β-catenin Signaling Pathway In Epithelial Ovarian Cancer

Golgi phosphoprotein 3(GOLPH3),is newly identified 34 k Da phosphorylated matrix protein,which localizes to the transface of Golgi complex and plays a critical role in the DNA damage and Golgi secretory pathway.Recent studies indicate that GOLPH3 correlates with poor prognosis and clinical stages in several types of tumors and is involved in cancer progression.However,its biological role in epithelial ovarian cancer(EOC)and underlying mechanism remain poorly understood.We are also interested in whether GOLPH3 is involved in invasion and metastasis of EOC.Here,we found that,compared with normal ovarian cell line(MOODY)and benign ovarian tumor tissues,GOLPH3 was overexpressed in EOC cell lines(SKOV3 and HEY)and tissues.Moreover,to examine the role of GOLPH3,we knocked down its expression in HEY and SKOV3 cells and overexpressed it in HO8910 cells.Using Transwell and scratch migration assays,we found that GOLPH3 promoted EOC cells the migration and invasion.Epithelial–mesenchymal transition(EMT)is defined as a process in invasion and metastasis of many cancers,which is regulated by some signaling pathways,such as Wnt/ β-catenin signaling pathway.Furthermore,GOLPH3 upregulated the expression level of EMT markers,such as N-cadherin and Snail,which were restored via silencing GOLPH3 expression.The inhibitor or activator of the Wnt/β-catenin pathway,XAV939 or Li Cl,decreased or enhanced,respectively,the effect of GOLPH3 on EMT.This suggested that GOLPH3 promoted EMT progression via activating Wnt/β-catenin signaling.In addition,we found that EDD,the DNA damage repair gene,was consistent with GOLPH3 in EOC cell lines and tissues,which also promoted the activated Wnt/β-catenin signaling and EMT process.These findings demonstrated that EDD might be a downstream factor of GOLPH3.Therefore,our findings confirm the existence of theGOLPH3–Wnt/β-catenin–EMT axis in EOC and GOLPH3 might be a new therapeutic target for epithelial ovarian cancer.Part Ⅰ.High GOLPH3 expression in epithelial ovarian cancerObjective:To detect the expression level of GOLPH3 in epithelial ovarian cancer tissues and cell lines.Methods:The expression level of GOLPH3 in benign ovarian tumor,borderline ovarian tumor and epithelial ovarian cancer tissues were detected by immunohistochemistry.The human ovarian cancer cell lines SKOV3,HEY,ES-2,HO8910,HO8910-PM and normal ovarian epithelial cell line MOODY were cultured in common method and their proteins and RNA were extracted respectively.The expression levels of GOLPH3 in different cell lines were assessed by Western Blot and q RT-PCR assays.Results:(1)GOLPH3 expression was higher in epithelial ovarian cancer than in the borderline tumors and benign cystadenomas,and its expression was mainly located in the cytoplasm.(2)The results of q RT-PCR and Western Blot assays showed that compared with normal ovarian epithelial cell MOODY,GOLPH3 was highly expressed in epithelial ovarian cancer cell lines SKOV3,HEY and HO8910-PM.In addition,SKOV3 and HEY had a higher expression level of GOLPH3 expression than the other cell lines,whereas HO8910 had the lowest expression.Conclusions:GOLPH3 was highly expressed in EOC.Part Ⅱ.GOLPH3 promotes EMT process of epithelial ovarian cancer cellsObjective:To investigate the role of GOLPH3 in the migration and invasion of epithelial ovarian cancer cells and the EMT progression.Methods:To examine the role of GOLPH3 in EOC,we silenced its expression in SKOV3 and HEY cells or overexpressed GOLPH3 in HO8910 cells by using si-RNA technique or overexpression plasmid.The transfection efficiency was detected by q RT-PCR and Western Blot.Transwell and Scratch migration assays were used to detect the invasion and migration ability of treated cell lines.The expression of EMT-related proteins,E-cadherin,N-cadherin and Snail,were detected by Western Blot.Results:(1)The results of Western Blot and q RT-PCR showed that the relative expression of GOLPH3 m RNA and protein was significantly decreased in the si RNA-transfected group and increased in the plasmid-treated group.(2)Transwell invasion and migration experiments showed that silencing GOLPH3 significantly reduced the number of cells on the membrane filters for control,while overexpressing GOLPH3 increased the number of cells on the membrane.(3)Scratch migration test results showed that silencing the GOLPH3 expression can decrease the wound healing ability of SKOV3 and HEY cells,which was in contrast to GOLPH3 overexpression in HO8910 cells.(4)Compared to the control group,knockdown of GOLPH3 expression in HEY and SKOV3 cells could inhibit the expression of E-cadherin,and stimulated the expression of N-cadherin and Snail,which was restored by increasing GOLPH3 expression in HO8910 cells.Conclusions:These results indicate that GOLPH3 contributes to the migration and invasion capacity and promotes EMT of EOC.Part Ⅲ.GOLPH3 induces EMT through activating the Wnt/β-catenin signaling pathwayObjective:To explore the underlying molecular mechanism of GOLPH3 in promoting EMT in epithelial ovarian cancer.Methods : We silenced GOLPH3 expression in SKOV3 and HEY cells or overexpressed GOLPH3 in HO8910 cells by using si-RNA technique or overexpression plasmid.Western Blot was used to detect the expression of Wnt/β-catenin signaling-related proteins,β-catenin,c-Myc and cyclin-D1.The inhibitor and activator of the Wnt/β-catenin signaling pathway,XAV939 and Li Cl,were used to treat GOLPHHEY and HO8910 cells respectively.The expression of Wnt / β-catenin pathway and EMT-related proteins were detected by Western Blot.Results:(1)Western Blot showed that GOLPH3 overexpression in HO8910 cells significantly increased the expression of β-catenin,c-Myc,and cyclin-D1.Knockdown of GOLPH3 in HEY and SKOV3 cells suppressed the expression of these genes.These results indicate that the Wnt/β-catenin signaling pathway plays a functional role in GOLPH3-induced EMT in EOC.(2)Western Blot showed that the EMT-related proteins were significantly increased or decreased after using Wnt / β-catenin signaling pathway agonist Li Cl or blocker XAV939,which indicated that the EMT process was enhanced or reversed.Conclusions:These data suggest that GOLPH3 can promote EMT by activating Wnt / β-catenin signaling pathway in epithelial ovarian cancer.Part Ⅳ.GOLPH3 might regulate EDD during Wnt signaling-induced EMT in EOC cellsObjective:To explore the relationship between EDD and GOLPH3,and to exam the role and mechanism of EDD in the process of GOLPH3 induced-EMT in epithelial ovarian cancer.Methods:Immunohistochemical technique was used to detect the expression level of GOLPH3 and EDD in the same sections of benign ovarian tumor,borderline ovarian tumor and epithelial ovarian cancer tissues.Western Blot and q RT-PCR were used to exam the expression of EDD in GOLPH3-overexpressed HEY cells or GOLPH3-silenced HO8910 cells.We silenced EDD expression in HEY cell line by using si-RNA technique.The expression levels of Wnt / β-catenin signaling pathway and EMT-related proteins were detected by Western Blot.Results:(1)We found that the expression levels of EDD were consistent with GOLPH3 in different ovarian tissues by using immunohistochemistry assays.GOLPH3 expression was mainly located in the cytoplasm,while EDD expression was mainly located in the nucleus.(2)Western Blot and q RT-PCR showed that the expression level of EDD was decreased in GOLPH3-silenced HEY cells,while it was significantly increased in GOLPH3-overexpressed HO8910.(3)The si-RNA technique was used to silence EDD expression in HEY cell line The results of Western Blot showed that knockdown of EDD expression could reduce the expression of E-cadherin,and increase the expression of N-cadherin,Snail and Wnt / β-catenin signaling pathway-related proteins,like β-catenin,C-Myc and cyclin-D1.Conclusions:These findings demonstrate that EDD might be a downstream factor of GOLPH3,which could play a role in GOLPH3 induced-EMT process.