CPEB4 Induces EMT Of Nasopharyngeal Carcinoma By Regulating β-catenin Translation Level

Part Ⅰ Objective: to construct NPC cell lines with CPEB4 gene silencing and CPEB4 gene overexpression,and to examine the effect of CPEB4 expression on the EMT phenotype of CNE-2 cells in nasopharyngeal carcinoma.Methods: CNE-2 cell lines with CPEB4 gene silencing and CPEB4 gene overexpression were constructed by infection lentivirus and divided into four groups: CPEB4 overexpression control group,CPEB4 overexpression group,CPEB4 silencing control group and CPEB4 silencing group.The corresponding expressions of CPEB4,β-catenin,E-cadherin and Vimentin were detected by Western blotting in each group after transfection.The migration ability of the four groups of cells was measured at 0h,24 h and 48 h.Transwell assays detected the invasiveness of four groups of cells at 24 h.Results: Western blotting results showed that compared with the CPEB4 overexpression control group,the expression levels of CPEB4,β-catenin and Vimentin were increased in the CPEB4 overexpression group,while e-cadherin was decreased in the CPEB4 overexpression group,showing significant differences(P < 0.05).Compared with the CPEB4 silencing control group,the expression levels of CPEB4,β-catenin and Vimentin were decreased in the CPEB4 silencing group,while E-cadherin was increased in the CPEB4 silencing group,showing significant differences(P < 0.05).The Wound Healing assay results showed that,compared with the CPEB4 overexpression control group,the over-expression group had faster healing speed and stronger migration ability over time,and the difference was significant(P < 0.05).Compared with the CPEB4 silencing control group,the healing rate of the silencing group was slower and the migration ability was worse over time,showing a significant difference(P < 0.05).The results of Transwell experiment indicated that,compared with the CPEB4 overexpression control group,the over-expression group had stronger invasiveness and the difference was significant(P < 0.05).Compared with the CPEB4 silencing control group,the invasion ability of the silencing group was weaker and the difference was significant(P < 0.05).Conclusion: 1)changes in CPEB4 expression can result in corresponding changes in the expression of β-catenin in CNE-2 cells;2)changes in CPEB4 expression can lead to corresponding changes in the expression of EMT-related proteins in CNE-2 cells(such as e-cadherin and Vimentin).3)the expression of CPEB4 can change the migration and invasion ability of CNE-2 cells.4)the expression of CPEB4 may affect the occurrence of EMT in nasopharyngeal carcinoma.PartⅡ Objective: To observe the localization of CPEB4 and β-catenin in nasopharyngeal carcinoma cell line CNE-2 and to verify the binding of CPEB4 to β-catenin.Methods:(1)Bioinformatics analysis: screening of CPEB4 downstream molecules;(2)cellular immunofluorescence: observation of the localization of CPEB4 and β-catenin in nasopharyngeal carcinoma cell line CNE-2;(3)RIP technology(RNA)Binding protein Immunoprecipitation Assay,RNA binding protein immunoprecipitation): verification of the binding of CPEB4 to β-catenin;Results: Bioinformatics analysis showed that β-catenin may be a downstream molecule of CPEB4.Cellular immunofluorescence results indicated that CPEB4 and β-catenin colocalized in the cytoplasm of CNE-2 cells,suggesting that both may be useful.RIP showed that CPEB4 binds to β-catennin.Conclusion: 1)CPEB4 and β-catenin are co-expressed in the cytoplasm,suggesting that both may be useful.2)In addition,CPEB4 can regulate its expression by binding to β-catennin.PartⅢObjective: To demonstrate that CPEB4 induces the development of EMT in nasopharyngeal carcinoma cells by regulating the level of β-catenin translation.Methods: CPEB4 overexpression group was added to two β-catenin inhibitors as experimental group,and CPEB4 overexpression group without any treatment was used as control group.Western blotting was used to detect the corresponding expression levels of β-catenin,E-cadherin and Vimentin in the three groups.Scratch test was used to detect the migration ability of the three groups of cells at 0h,24 h and 48 h.Transwell assay was used to detect the invasive ability of the three groups of cells at 24 h.Results: Western blotting showed that the expression level of E-cadherin was increased in the two groups,and the expression levels of β-catenin and Vimentin were decreased in the two groups(P<0.05).).The results of the scratch test showed that the healing rate of the two groups was slower and the migration ability was worse than that of the control group,and the difference was significant(P<0.05).Transwell results showed that the invasive ability of the two groups was weaker than the control group,and the difference was significant(P<0.05).Conclusion: The experiment confirmed that CPEB4 can induce the development of EMT in nasopharyngeal carcinoma cells by regulating the level of β-catenin translation.