| Nasopharynx cancer is a high incidence of cancer in the south of China and Southeast Asian countries which contributes a great threat to humans' health.The high rate of neoplasm recurrence and distant metastasis contribute to a high rate of death and failure of therapy.Recently,it is found that cancer stem cells exist in many malignant tumors and make cancers,including nasopharynx cancer,show a high rate of neoplasm recurrence and resistance to radiotherapy and chemicals.Cancer stem cells show strong abilities of self-replication and high telomerase activity with biomarkers such as CD133 and CD44.Searching the mechanisms of development of cancer stem cells can provide a new method to cure malignant tumors.In the previous study in our laboratory,PinXl is found to inhibit nasopharynx cancer as a telomerase inhibitor.In this study,we detect the effected of PinXl in CD 133+ cancer stem cells,including the inhibition of growth,migration and the telomerase activity of cancer stem cells.Furthermore,the mechanism of telomerase inhibition on TRF1 and TRF2 related was detected,indicating that PinXl could be used as a target of drug development on cancer stem cells.Part I CD133+ cells of nasopharynx cancer showed characters of cancer stem cellsObjective:This study is aimed to sort CD133+ cancer stem cells and observe the abilities of proliferation and migration abilities of CD133+ cells.Methods:CD133+ and CD133-cells of nasopharynx cancer C:NE2 cells were sorted through magnetic activated cell sorting methods and were validated through flow cytometry assays.Then CD133+ and CD133-cells were incubated with stem cell induced medium to form stem cell spheres.The cells were planted in nude mice to observe the cancer proliferation.Nanog and SOX2 expression were detected through QPCR assays.Then the proliferation of these cells was observed through CCK-8 eassays and the transwell assay and scoring assay were taken to explore the migration and invasion abilities.Results:1.CD133 positive cells and negative cells were sorted successfully through magnetic activated cell sorting at 3 days after sorting.2.The sorted cells were incubated with culture medium contained bFGF,EGF,insulin and B27 free of serum for 14 days.It was found that the stem cell spheres semi diameters of CD133+ were larger than CD133-cells,suggested that the cancer stem cells were sorted successfully.3.The expression of Nanog and SOX2 in CD133+ cells were more than CD133-cells by QPCR assay,suggested that CD 133+ cells have the same characters to stem cells.4.The rate of proliferation of CD133+ was higher than CD133-cells significantly(P<0.05)detected by CCK-8 assay.5.The migration and invasion of CD133+ and CD133-cells were detected through transwell assays or scoring assays,and the migration and invasion of CD133+cells is more than CD133-cells significantly(P<0.05).6.CD 133+ cells showed a higher proliferation than CD 133-cells in nude mouse transplantation tumor experiment.Conclusion:In this part,CD133+ cells and CD133-cells were sorted through magnetic activated cell sorting.CD133+ cells showed stronger abilities of proliferation,migration and invasion,indicating that CD 133 is a potent marker of nasopharynx cancer stem cells.Part ? PinX1 targets telomerase and inhibits the stem cell activity of nasopharynx cancer stem cellsObjective:This part is aimed to detect the influence of PinX1 on the stem cell activity of nasopharynx cancer stem cells.Methods:CD 133+ cells were transfected with PinX1 overexpression plasmid or vector and CD133-cells were transfected with PinX1 siRNA and NC siRNA.Then cells were incubated with stem cell induced medium to form stem cell spheres.The proliferation of these cells was observed through CCK-8 assays and the transwell assay and scoring assay were taken to explore the migration and invasion abilities.The apoptosis of cells were examined through flow cytometry assays.Results:1.The expression of hTERT in CD133+ cells was higher than CD133-cells,indicating that the telomerase activity in CD133+ is strong than CD133-cells.The expression of PINX1 in CD133+ cells was less than CD133-cells.2.CD 133+ cells transfected with PINX1 overexpression plasmid increased lower than vector group.CD133-cells transfected with PINX1 siRNA increased higher than NC siRNA.3.These cells were Stem cell spheres semi diameters of CD133+ transfected with PinX1 overexpression plasmid was smaller than cells transfected with vector.The stem cell spheres semi diameters of CD133-transfected with PinX1 siRNA was larger than cells transfected with NC siRNA after incubating with culture medium contained bFGF,EGF,insulin and B27 free of serum for 14 days.4.Trough scoring assays and transwell assays,it was found that PinX1 could inhibit the migration and invasion of CDD133+ cells.5.The apoptsis rate of CD133+ cells transfected with PinX1 overexpression plasmid increased and CD 133-cells transfected with PinX1 siRNA decreased.Conclusion:PinX1 could inhibit the proliferation,migration and invasion of CD 133+cells,indicating that PinX1 inhibits the stem cell activity of nasopharynx cancer stem cellsPart ? Telomerase and TRFs in the inhibition of PinXl on nasopharynx cancer stem cellsObjective:To detect the telomerase-TRFs in the PinXl regulation on nasopharynx cancer stem cells.Methods:The telomerase activity marker,hTERT,was deteted by QPCR as well as PinXl.PinXl overexpression plasmid and siRNA were built successfully.CD133+cells were transfected with PINX1 overexpression plasmid or the vector while CD133-cells were transfected with PinXl siRNA or NC siRNA.Further,QPCR and WB assays were taken to observe the expression of hTERT,PinXl and TRFs.Results:1.The expression of hTERT in CD133+ cells was higher than CD 133-cells and PinXl was lower in CD133+ cells,indicating that the telomerase activity in CD133+ is strong than CD133-cells.2.PinXl overexpression plasmid and siRNA were built successfully and validated through QPCR and WB assays transfected in CNE2 cells.3.The mRNA and protein level of hTERT decreased when CD 133+ cells transfected with PinXl plasmid,and increased when CD 133-transfected with PinX1 siRNA,while TRF1 showed an opposite trend and TRF2 didn' t change.Conclusion:In this part,we found that PinXl could suppress telomerase in nasopharynx cancer stem cells and downregulate TRF1 expression.In other words,PinXl could inhibit nasopharynx cancer stem cells through binding telomerase-TRF1. |
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