PinX1 Inhibits EMT Of Nasopharyngeal Carcinoma Stem Cells Through Targeting Telomerase

Nasopharyngeal carcinoma is one of the common malignant tumors in southern China and Southeast Asia,and it is called "Guangdong tumor".Recurrence and metastasis are the main causes of treatment failure in nasopharyngeal carcinoma.Cancer stem cells are considered to be the source of tumor proliferation,metastasis and recurrence.Telomerase is highly expressed in most tumors including nasopharyngeal carcinoma.PinX1 is the major telomerase inhibitor.Epithelial mesenchymal transition(EMT)is a common biological process of tumor metastasis.The relationship between tumor stem cells,telomerase and EMT in nasopharyngeal carcinoma is still unclear.Therefore,it is important to use telomerase inhibitors to target telomerase activity to inhibit the expression of tumor stem cells and EMT.PART Ⅰ Sorting and biological characteristics of CD133+nasopharyngeal carcinoma stem cellsObjective:To investigate the biological characteristics of CD133+ cells sorted by CD 133 as a stem cell marker.Methods:CNE2 cells were sorted into CD133+ and CD133-cells by magnetic bead sorting.The efficiency of sorting was verified by flow cytometry and fluorescence immunoassay.Cells were detected by in vitro globule formation,CCK-8 and Transwell assay.In vitro globule,cell proliferation,invasion,migration ability.Result:1.The unlabeled CNE2 cells were used as control.Flow cytometry and fluorescence immunoassay were performed on the cells after magnetic bead sorting.The results showed that CD133+ cells were successfully sorted.2.The sorted cells were induced to culture in vitro.After 10 days,the radii of the tumor cells formea by CD 133+ cells were significantly larger than those of CD 133-cells,and the number of globules was more.3.The proliferation rate,migration and invasion ability of CD133+ cells were significantly higher than those of CD133-cells.Conclusion:CNE2 cells of nasopharyngeal carcinoma can successfully obtain CD 133+nasopharyngeal carcinoma stem cells by magnetic bead sorting.At the same time,CD 133+ nasopharyngeal carcinoma stem cells have stronger characteristics of tumor stem cells than CD133-nasopharynx in vitro.PART Ⅱ PinX1 gene targeting telomerase inhibits EMT of nasopharyngeal carcinoma stem cellsObjective:To investigate the effect of PinX1 gene targeting telomerase on the biological characteristics and EMT of nasopharyngeal carcinoma stem cells.Methods:The expression of EMT in CD133+ and CD133-cells was detected by QPCR and Western blot.The activity of transfected PinX1 overexpression plasmid and corresponding empty vector plasmid in CD133+ cells was detected by QPCR and Western blot.The cells obtained by transfection were induced into stem cells by stem cells.The growth and proliferation of the cells were observed by CCK-8 method.The invasion and migration of the cells were detected by Transwell method.The EMT markers of nasopharyngeal carcinoma stem cells were detected by QPCR and Western blot.,the expression of transcriptional regulatory factors.Result:1.The expression of EMT markers in nasopharyngeal carcinoma was detected.The E-cadherin of CD133+ nasopharyngeal carcinoma stem cell epithelial markers was significantly lower than that of CD133-cells.The mesenchymal marker Vimentin was significantly higher than CD133-cells.2.When CD133+ cells were transfected with PinX1 overexpression plasmid,telomerase activity was significantly inhibited.In terms of stem cell biological characteristics,cell glomerizing ability was significantly weakened,stem cell sphere radius and number of pellets were significantly reduced;cell proliferation,The ability to invade and migrate was significantly lower than that of the empty plasmid group.3.After transfection of CD133+ cells with PinX1 overexpression plasmid,the expression of E-cadherin was significantly higher than that of the empty plasmid group.The expression of Vimentin was significantly lower than that of the empty plasmid group.The expression of EMT transcriptional regulators Snail,Twist and Zeb1 was significantly lower than that of no-load.Plasmid group.Conclusion:1.CD133+ nasopharyngeal carcinoma stem cells exhibit stronger epithelial mesenchymal transition(EMT)potential than CD133-nasopharyngeal carcinoma cells;2.PinX1 gene can inhibit the telomerase activity of CD 133+ nasopharyngeal carcinoma stem cells,thereby inhibiting the biological characteristics of nasopharyngeal carcinoma stem cells and inhibiting the EMT potential.