Research On The Functional Difference And Its Mechanism Between Cervical Cancer Stem Cells And Cervical Cancer Cells

BackgroundCervical cancer is one of the main factors that endangers women’s health,especially those with poor prognosis such as metastasis and recurrence in advanced cervical cancer or refractory cervical cancer.Cancer stem cells have been considered as seed cells for tumor metastasis and recurrence.Therefore,laying stress on cancer stem cells,studying the functional differences and the related mechanisms between cervical cancer stem cells and ordinary cancer cells will help solving the treatment problems of advanced or refractory cervical cancer fundamentally.Purposes1.To identify the biological function differences between cervical cancer stem cells and cervical cancer cells.2.To clarify the difference in radiotherapy and chemotherapy resistance of cervical cancer stem cells and cervical cancer cells.3.To explore the mechanism by which SFRP1 regulates the maintenance of sternness of cervical cancer cells through the Wnt/β-catenin pathway.Method1.First,isolate the side population stem cells(SP cells)and non-side population stem cells(NSP cells)from human SiHa cervical cancer cell lines by flow cytometry,and then after culturing and passaging,CCK8,Edu,transvell,plate clone formation,sphere assays were used to detection of the differences in proliferation,migration,cloning and sphere abilities between the SP cells and NSP cells.At the same time,the subcutaneous tumor formation ability of the cells was observed through the subcutaneous transplantation tumor experiment in nude mice.The expression of the sternness markers(CD 133,CD44 and Nanog)of the cells were detected by western blot.2.After paclitaxel,cisplatin therapy or radiotherapy,CCK8,Edu,transvell,plate clone formation assays were used to detect the proliferation,migration,cloning abilities of the cells.The expression of the stemness markers(CD 133,CD44 and Nanog)of the cells were detected by western blot.3.After transcriptome sequencing,the differentially expressed genes and functional enrichment analysis of cervical cancer stem cells and cervical cancer cells were performed.Western blot was used to test the expression of SFRP1 and the key proteins of the Wnt/β-catenin pathway(β-catenin,C-myc,Cyclin D1),then immunofluorescence was used to detect the intracellular expression of β-catenin and ELISA to detect the secretion of SFRP1 by the cells.Results1.The level of stemness markers and the abilities of proliferation,migration,cloning,sphere and subcutaneous tumor formation of SP cells sorted by flow cytometry are significantly stronger than NSP cells.2.After paclitaxel,cisplatin or radiation treatment,the expression level of stemness markers and the abilities of proliferation,migration,and cloning of SP cells are still significantly stronger than NSP cells.3.The level of SFRP1 protein in cervical cancer stem cells is significantly higher than that in cervical cancer cells,but the secretion level is decreased.The Wnt/β-catenin pathway expression in cervical cancer stem cells is stronger than that in cervical cancer cells.Conclusion1.The SP cells have the function of cervical cancer stem cells,and are more resistant to radiotherapy and chemotherapy than NSP cells.Therefore,the SP cells sorted by flow cytometry can be used as a reliable cell model for cervical cancer stem cell research.2.Cervical cancer stem cells may inhibit the secretion of SFRP1,activate the Wnt/β-catenin pathway to regulate the stemness level and the resistance to radiotherapy and chemotherapy of cervical cancer stem cells,providing ideas for the development of targeted drugs for cervical cancer stem cells.