Role Of ATF5 In Invasion And Metastasis Of Nasopharyngeal Carcinoma And Its Mechanism

Objective: To investigate the effect of activating transcription factor 5(ATF5)on the invasion and migration ability of nasopharyngeal carcinoma cells and its possible molecular mechanism.Methods: Human nasopharyngeal carcinoma CNE2 Z cells were used as research objects,and lentiviral vector sh RNA-ATF5 interference technology was used to silence ATF5 expression of CNE2 Z cells and the interference efficiency of sh RNA-ATF5 carrier was detected by real-time fluorescent quantitative polymerase chain reaction(q RT-PCR).CNE2 Z cells not infected with lentivirus were used as blank control(Control group),and CNE2 Z cells infected with lentiviral empty vector LV-sh RNA-NC were used as negative control(NC group).LV-sh RNA-ATF5 infected CNE2 Z cells were the experimental group(sh RNA-ATF5 group).Next,We used MTT and clone formation test to detect CNE2 Z cell proliferation ability,scratch test,Transwell invasion test to detect CNE2 Z cell invasion and migration ability.Affymetrix gene expression profiling chip technology was used to compare gene expression changes in CNE2 Z cells after infection with sh RNA-ATF5 lentivirus and Ingenuity pathway analysis(IPA)enrichment of ATF5-related integrin-linked kinase(Integrin linked kinase,ILK)signal pathway,and analysis of E-cadherin,N-cadherin,matrix metalloproteinase 9(MMP9),Viminetine after CNE2 Z cells silence ATF5 by Western blotting,as well as changes in ILK and Glycogen synthase kinase 3?(GSK3?)expression levels,and then observed the effect of ATF5 over-expression on the expression levels of ILK and GSK3? in CNE2 Z cells.Results:(1)Both q RT-PCR and Western blot results showed that sh RNA-ATF5 lentiviral vector could significantly inhibit the expression of ATF5 in nasopharyngeal carcinoma CNE2 Z cells(P <0.05),and the interference efficiency could reach 91.2%.(2)MTT test results showed that CNE2 Z cells in the sh RNA-ATF5 group had significantly lower proliferation than the NC group(P<0.05);the results of the colony formation experiments also revealed that compared with the number of clones of CNE2 Z cells in the NC group(106 ± 3.21),the sh RNA-ATF5 group cells The number of clones(10 ± 2.08)was significantly reduced(P <0.05).(3)The scratch test results showed that the CNE2 Z cells in the NC group had a migration rate of 0.52 ± 0.05 at 4hours and a migration rate of 0.74 ± 0.07 at 8 hours.Cell migration in the sh RNA-ATF5 group was 0.17 ± 0.04 at 4 hours and 0.18 ± 0.06 at 8 hours.The cell migration capacity of the sh RNA-ATF5 group was significantly lower than that of the NC group(P <0.05).The number of migrating cells in the sh RNA-ATF5 group(2 ±0.23)was significantly lower than that of NC group(111 ± 1.99)(P <0.05);the results of the Transwell invasion experiment also showed that the number of invading cells in the sh RNA-ATF5 group(4 ± 1.45)was significantly lower Number of invasive cells in the NC group(132 ± 9.79)(P <0.05)(4)IPA analysis results show that ATF5-related downstream pathways contained HGF signal pathway,TGF-? signal pathway,signal pathway,IL-6 signal pathway,ILK signal pathway,etc.The ILK signal pathway was most significantly suppressed,and the Z-score was-2.111.(5)Westorn blot analysis showed that the expression of E-cadherin in CNE2 Z cells in the sh RNA-ATF5 group was significantly higher than that in the Control and NC groups,while the expression of N-cadherin,Viminetine,and MMP9 was significantly reduced(P<0.05).CNE2 Z in the sh RNA-ATF5 group The expression levels of ILK and p-GSK3?(Ser-9)in cells were significantly lower than those in Control group and NC group(P<0.05),while the expression levels of ILK and p-GSK3?(Ser-9)in CNE2 Z cells in ATF5 over-expression group were significantly higher than those in Control group and NC group(P<0.05)and there was no significant change in total GSK3? expression level(P>0.05).Conclusion:(1)ATF5 silencing could inhibit the proliferation of nasopharyngeal carcinoma cells,reduce the ability of invasion and migration of nasopharyngeal carcinoma cells.(2)ATF5 silencing could up-regulate the expression of E-cadherin protein and down-regulate the expression of N-cadherin,Vimentin and MMP9,which suggesting that ATF5 silencing can inhibit or block the EMT of nasopharyngeal carcinoma cells.(3)ATF5 silencing may weaken the invasion and migration ability of nasopharyngeal carcinoma cells by inhibiting the ILK/GSK? signaling pathway to block the EMT of nasopharyngeal carcinoma cells.