Regulation Of Notch1 On Invasion And Migration Of Cisplatin-resistant Nasopharyngeal Carcinoma Cells

Aims: 1.To test whether the resistance to cisplatin(DDP)of nasopharyngeal carcinoma cell line and the invasion and migration ability of nasopharyngeal carcinoma cells is changed.To study whether DDP-resistant nasopharyngeal carcinoma cells has undergone epithelial-mesenchymal transition(EMT).2.To explore the changes of Notch1 in DDP-resistant nasopharyngeal carcinoma cells and to further study its relationship with EMT.3.To investigate the effect of Notch1 down-regulation on the invasion and migration of DDP-resistant nasopharyngeal carcinoma cells.4.The molecules that may be regulated by Notch1 were investigated in nasopharyngeal carcinoma DDP-resistant cell lines.Methods: 1.Concentration-increasing method was used to establish DDP-resistant nasopharyngeal carcinoma cell line,1.5 mmol/L DDP was used as the starting concentration,and the concentration was gradually increased according to the ability of cell proliferation.CNE-2Z cells were continuously exposed to DDP> 10 months in order to produce stable DDP-resistant.nasopharyngeal carcinoma cells.2.The DDP-resistance of nasopharyngeal carcinoma cells was tested by MTT assay.3.The migration and invasion ability of CNE-2Z,CNE-2Z/DDP,HNE-1 and HNE-1/DDP cells were detected by cell scratch test and Transwell chamber assay.The morphological characteristics of CNE-2Z/DDP,HNE-1 and HNE-1/DDP cells were observed under inverted microscope.And the expression of EMT-related proteins,such as E-cadherin,Vimentin and MMP-9 was analyzed by Western blot.4.The Notch1 si RNA was transfected into DDP-resistant nasopharyngeal carcinoma cells.The change of Notch1 protein was detected by Western blot.The cell migration ability was detected by cell scratches.The invasion and migration ability of the cells were detected by Transwell chamber assay.The change of EMT-related proteins was detected by Western blot.Results: 1.Verification of the resistance to DDP of CNE-2Z / DDP and HNE-1 / DDP cells MTT assay was used to detect the survival rate followed exposure to DDP(2,4,8,16,32 μmol/L)in four nasopharyngeal carcinoma cells CNE-2Z,CNE-2Z/DDP,HNE-1,HNE-1/DDP for 24,48,72 h.The resistance index(RI)of HNE-1/DDP cells to HNE1 cells was 4.26,4.05,4.79 at 24,48 and 72 h,respectively.The RI of CNE-2Z/DDP cells to CNE-2Z cells was 3.07,3.10,3.32 at 24,48 and 72 h,respectively.2.DDP-resistant nasopharyngeal carcinoma cells exhited stronger invasion and migration bility and EMT characteristics(1)The results of wound healing assay showed that CNE-2Z/DDP and HNE-1/DDP cells hadstronger migration ability compared with CNE-2Z and HNE1 cells.The results of Transwell chamber assay showed that the migration and invasion ability of CNE-2Z/DDP,HNE1/DDP cells in vitro were significantly enhanced.compared with CNE-2Z and HNE-1 cells(2)The morphological characteristics of CNE-2Z/DDP and HNE-1/DDP were observed under microscope,and the morphological changes were observed in the DDP-resistant cells compared with the parental cell lines.Resistant cells CNE-2Z/DDP and HNE-1/DDP cells were similar to interstitial changes,including cell polarity loss and increased pseudopodia formation,cell-to-matrix connectivity,elongation,and dispersal growth,irregular the appearance of fibroblasts.(3)Western blot showed that the expression of epithelial markers in cisplatin-resistant nasopharyngeal carcinoma cells was significantly lower than that in parental cells,while the expression of interstitial markers(such as Vimentin,MMP-9)in resistant nasopharyngeal carcinoma cells was significantly higher than that in parent cells.3.Expression of Notch1 in nasopharyngeal carcinoma cells Western blot showed that Notch1 expression was up-regulated in CNE-2Z/DDP and HNE-1/DDP cells compared with CNE-2Z and HNE-1 cells.4.Down-regulation of Notch1 on invasion and migration of DDP-resistant nasopharyngeal carcinoma cells(1)Transfection of Notch1 si RNA and control si RNA into DDP-resistant nasopharyngeal carcinoma cells CNE-2Z/DDP,HNE-1/DDP,Western blot was applied to verify the transfection results.The expression of Notch1 was down-regulated,indicating interference success.(2)The results of wound healing assay showed that transfection of Notch1 si RNA could significantly inhibit the migration ability of CNE-2Z/DDP and HNE-1/DDP cells.In addition,Transwell chamber results showed that transfection of Notch1 si RNA could significantly reduce the invasion ability of CNE-2Z/DDP,HNE1/DDP cells.(3)Western blot results showed that transfection of Notch1 si RNA resulted in an increase in E-cadherin and reduction of Vimentin and MMP-9 in CNE-2Z/DDP,HNE-1/DDP cells.Notch1 si RNA could transform CNE-2Z/DDP,HNE1/DDP cells from interstitial spindle cells into epithelioid cells.5.Different expression of GRP 78 and CHOP in nasopharyngeal carcinoma cells and up-regulation of Notch1 on the expression of CHOP(1)GRP 78 had the same expression in CNE-2Z/DDP and CNE-2Z cells.The expression of GRP 78 in HNE-1/DDP cells was down-regulated compared with HNE-1 cells.While CHOP expresion in CNE-2Z,HNE-1 cells was significantly higher than that in CNE-2Z/DDP and HNE-1/DDP cells.(2)Down-regulation of CHOP was observed after Notch1 over-expression in nasopharyngeal carcinoma CNE-2Z and HNE-1 cells.Conclusion: 1.Nasopharyngeal carcinoma CNE-2Z/DDP and HNE-1/DDP cells is resistant to DDP.2.The invasion and migration capacity is increased in CNE-2Z/DDP and HNE-1/DDP cells.3.Notch1 is up-regulated in DDP-resistant nasopharyngeal carcinoma cells and may be involved in DDP-resistance and EMT.4.Down-regulation of Notch1 can inhibit the invasion and migration of DDPresistant nasopharyngeal carcinoma cells,and reverse EMT.