LGR5 Promotes Epithelial Ovarian Cancer Proliferation,metastasis,and Epithelial-mesenchymal Transition Through The Notch1 Signaling Pathway

Part Ⅰ The expression of LGR5 mRNA in ovarian cancer tissue in Oncomine databaseObjective:Clarify the role of LGR5 in EOC development and its mechanism,we first reviewed the expression of LGR5 mRNA in ovarian cancer tissue in the database of Oncomine(www.oncomine.org).Methods:We first reviewed the expression of LGR5 mRNA in ovarian cancer tissue in the database of Oncomine(www.oncomine.org),the relevant data were sorted out.Results:(1)compared with normal ovarian tissue,LGR5 mRNA expression increased in ovarian cancer.(2)LGR5 mRNA expression level increased in Grade2-3 of ovarian cancer.(3)LGR5 mRNA expression increased in stage Ⅲ or Ⅳ ovarian cancer.Conclusions:The expression of LGR5 mRNA in ovarian cancer tissues was elevated in Oncomine database.Part Ⅱ LGR5 is overexpressed in EOC tumor tissues and some cell linesObjective:In order to further study the function of LGR5 in epithelial ovarian cancer and its mechanism,we detected the expression of LGR5 in epithelial ovarian cancer cell lines and EOC tumor tissues and analyzed clinicopathologic correlation.Methods:Immunohistochemical method was used to detect the expression of LGR5 protein in epithelial ovarian cancer tissue section.The expression levels of LGR5 in cell line SKOV3,HEY,HO8910 and Moody were detected by Western blot and RT-PCR.Results:(1)The expression level of LGR5 in epithelial ovarian cancer tissues was significantly higher than that of normal ovarian tissue.(2)The expression level of LGR5 was correlated with metastasis(P=0.025),age(P=0.026)and pathological type(P<0.001).(3)The mRNA and protein level of LGR5 in HEY,SKOV3,HO8910 were higher than the control cell line--Moody.Conclusions:LGR5 is overexpressed in EOC tumor tissues and some cell linesPart Ⅲ LGR5 could enhance the proliferation,invasion and metastasis capacity of epithelial ovarian cancer cellsObjective:In order to explore the effect of LGR5 on the proliferation,invasion and metastasis capacity of epithelial ovarian carcinoma cells in vitroMethods:Small interfering RNA(siRNA)was transfected into SKOV3 or Hey cells to silence LGR5 expression and the LGR5 plasmid was transfected into HO8910 to upregulate the expression level of LGR5.The effect of LGR5 expression level on the proliferation ability of epithelial ovarian cancer cells was investigated by using CCK-8 kit and colony formation assay.Transwell invasion assay and cell scratch-wound assay were applied to detect the effect of LGR5 on invasion and metastasis capacity of epithelial ovarian cancer cells.Results:(1)The upregulation of LGR5 could enhance the proliferation capacity of epithelial ovarian cancer cells.(2)The upregulation of LGR5 could enhance the invasion and metastasis capacity of epithelial ovarian cancer cell.Conclusion:LGR5 could enhance the proliferation,invasion and metastasis capacity of epithelial ovarian cancer cells.Part Ⅳ LGR5 could induce epithelial-mesenchymal transformation in epithelial ovarian cancerObjective:To explore the role of LGR5 in the process of epithelial ovarian epithelial mesenchymal transformation(EMT)in epithelial ovarian carcinomaMethods:Western blot was used to detect the influence of LGR5 on EMT related proteins,such as epithelial marker protein E-cadherin,the mesenchymal marker N-cadherin,Vimentin,and EMT related transcription factor Snail.Results:(1)Downregulation of LGR5 expression could reduce the expression level of the mesenchymal marker N-cadherin,Vimentin and EMT related transcription factors,and increase the expression level of epithelial marker,E-cadherin.(2)Increasing the expression of LGR5 could upregulate the expression level of N-cadherin,Vimentin and EMT related expression level of transcription factors,at the same time reduce the epithelial symbol E-cadherin protein expression level.Conclusions:LGR5 could induce epithelial-mesenchymal transformation in epithelial ovarian carcinoma.Part Ⅴ LGR5 could regulate the Notch1 pathway in epithelial ovarian cancerObjective:To explore the relationship between LGR5 and Notch1 pathway in epithelial ovarian cancer,and further explore the mechanism of LGR5 effect on the progression of epithelial ovarian cancer.Methods:After downregulation or overexpression of LGR5 in epithelial ovarian cancer cell lines SKOV3 and HO8910,Western blot was used to detect the effect of LGR5 on the expression of Notch1 and its downstream target gene HES1.In order to further explore the relationship between LGR5 and Notch1 signaling pathway,the Notchl pathway inhibitor DAPT was applied in HO8910 and analyzed by Western blot.Results:(1)After the knockdown of LGR5 in SKOV3,the activity of Notchl pathway can be inhibited.(2)DAPT-mediated downregulation of HES1 could be restored by overexpressing LGR5.Conclusions:LGR5 could regulate the Notch1 pathway in epithelial ovarian cancer.Part VI LGR5 promotes tumorigenesis in vivoObjective:To clarify the effect of LGR5 on the progression of epithelial ovarian cancer in vivo.Methods:After injection of control SKOV3 cells and and KD-LGR5 SKOV3 cells into nude mice,the volume and weight of the subcutaneous tumors were measured after the tumors grew.Results:(1)Tumors formed by injection of cells treated with LGR5-KD were significantly smaller than those treated with scrambled control.Tumor growth and tumor weight were significantly inhibited in LGR5-KD injected tumors.Conclusions:LGR5 promotes tumorigenesis in vivo.