Effects Of Silenced ATF5 On Apoptosis And Cell Cycle Of Nasopharyngeal Carcinoma

Objective: To compare the changes of apoptosis and cell cycles progression of nasopharyngeal carcinoma CNE-2Z cells with or without silencing transcription factor 5(ATF5),and explore the effects of ATF5 silencing on apoptosis and cell cycles of nasopharyngeal carcinoma cells and their possible mechanisms,providing new ideas for improving the therapeutic effect of nasopharyngeal carcinoma.Methods: 1)Using nasopharyngeal carcinoma CNE-2Z cells as the research object,the constructed recombinant lentiviral vector targeting sh RNA of ATF5 gene was infected into CNE-2Z cells(sh RNA-ATF5 group),Lentivirus empty vector was used to infect CNE-2Z cells(LV-sh RNA-NC)as a negative control group(NC group),uninfected lentivirus CNE-2Z cells were used as a blank control group(Control group).2)The transfection efficiency was evaluated by q RT-PCR method and Western blot technology.3)Flow cytometry with the Annexin V-APC single staining method and Tunel immunofluorescence test were applied on detecting the changes of the apoptosis in CNE-2Z cells with or without ATF5 silencing.PI-FACS method of the flow cytometry was also used to distinguish the discrepancy of progressions about cell cycle with or without ATF5 silencing.4)Western blot detection on the expression levels of CNE-2Z apoptosis-related proteins Bcl-2,Bax,and cyclin including Cyclin D1,cyclin-dependent kinase 4(CDK4),cell division control protein kinase 2(Cdc2)and Cyclin B1 with ATF5 silencing and overexpression.Results: 1)Detection of q RT-PCR and Western blot both showed that sh RNA-ATF5 lentiviral vector can significantly inhibit the expression of ATF5 in nasopharyngeal carcinoma CNE-2Z cells with 81.7% for interference efficiency(P<0.01);(2)Flow cytometry instrumental analysis showed that the apoptosis rates of CNE-2Z cells in the Control group,NC group,and sh RNA-ATF5 group were respectively 3.61(4)± 0.10,3.80(4)± 0.18,6.26(4)± 0.15.Sh RNA-ATF5 could increase the apoptosis rate of CNE-2Z cells significantly(P(27)0.01);Tunel experiment also revealed that the staining intensity of CNE-2Z cells in the sh RNA-ATF5 group was significantly increased compared to the Control group and NC group.(3)The results of PI-FACS analysis showed that the number of CNE-2Z cells in the G1 phase was significantly reduced in sh RNA-ATF5 group(P<0.01),while the number of cells in the S phase and G2/M phase increased,with G2/M phase more significantly(P<0.05,P<0.01).(4)Western blot analysis showed that CNE-2Z cells in the sh RNA-ATF5 group had significantly reduced the expression level of Bcl-2 protein and improved the expression level of Bax protein compared to the Control group and NC group(P<0.01).Cyclin D1 and CDK4 proteins,as regulatory proteins in the G1/S phase;Cdc2 and Cyclin B1,as regulatory proteins in G2/M phase,were all significantly reduced(all P<0.01).(5)Western Blot results dictated that of Bcl-2 protein expression in CNE-2Z cells with ATF5 overexpression was significantly increased(P<0.01),while Bax protein expression was significantly decreased(P<0.01);and the expression levels of G1/S phase regulatory proteins Cyclin D1,CDK4 and G2/M phase regulatory proteins Cdc2,Cyclin B1 were all significantly increased(all P<0.05).Conclusions:(1)ATF5 silencing can promote apoptosis of nasopharyngeal carcinoma CNE-2Z cells,which may be partly ascribed to increasing the expression of Bax protein and decreasing the expression of Bcl-2 protein;(2)ATF5 silencing can block cell cycles of CNE-2Z cells in G2/M phase,which may be partly due to the down-regulation of Cyclin D1/CDK4 and Cdc2/Cyclin B1 protein expression.