Research On Effects Of IKBKE On Migration And Invasion Of GBM Cells By Regulating Transcription Factor Snail1

Glioblastoma multiform(GBM)is a common malignant neuroepithelial tumor of the central nervous system in adults.It is invasive,leading to high recurrence rate,poor prognosis and complicated treatment process.The exploration of the pathogenesis and treatment of glioblastoma has been a hot topic in tumor research.Many studies have shown that IKBKE,also known as IKK? or IKKi,is a new member of the I?B kinase family,which regulates the interferon and nuclear transcription factor B pathway.Recently,studies have shown that IKBKE is related to the occurrence and progression of a variety of tumors and it is considered as a carcinogenic protein,which regulates the proliferation,migration and invasion of glioma cells.IKBKE plays an oncogene role in the growth and progression of tumors.Snail1 is a zinc finger protein transcription factor that inhibits the expression of E-cadherin in epithelial cells at the transcriptional level.The stable expression of Snail1 can lead to the loss of E-cadherin expression and epithelial interstitial change(EMT),thus leading to the occurrence,development,migration and invasion of glioma cells.In this paper,the purpose of our study is to explore whether IKBKE regulate the expression level of Snail1 and E-cadherin,and then explore the existence and function of IKBKE-Snail1-E-cadherin pathways,and explore the molecular mechanism of IKBKE mediated Snail1 stability.In this study,the expression of IKBKE and Snail1 in brain glioma specimens and glioblastoma cell lines and the interaction between them were investigated by immunohistochemical staining and western blot.This study mainly explored the interaction between IKBKE and Snail1 through co-immunoprecipitation(co-ip)and western-blot(WB),and further explore the stable molecular mechanism of IKBKE mediated Snail1 and its influence on the invasion and migration of glioma cells.MethodThe first part is a preliminary study on the effect of IKBKE on tumor invasion and migration and its correlation with Snail1.1.IKBKE overexpression vector and IKBKE-shRNA lentivirus were constructed.IKBKE overexpression plasmid was transfected into U118 glioma cell lines to upregulate the expression level of IKBKE in glioma cells.U87 and LN229 glioma cell lines were transfected with IKBKE-shRNA lentivirus to knock down the expression level of IKBKE in glioma cells.The effect of IKBKE on the invasion and migration of glioblastoma cells was detected by scratch test and Transwell chamber method,respectively.2.The expression of IKBKE and Snail1 in GBM cell lines(A172,U251,LN18,LN229,U118,U87,SNB19 and LN308)was detected by western blot method and the correlation was analyzed.3.U87 and LN229 cells were selected and transfected with IKBKE-shRNA lentivirus vectors in vitro.The effect of knockdown IKBKE gene on the expression levels of Snail1 and E-cadherin was detected by western blotting method,The effects of knocking down IKBKE gene on the expression of Snail1 protein and E-cadherin protein were detected by Western blotting.Western blotting method and immunofluorescence assay was used to verify the changes of snail1 protein content distribution in cytoplasm and nucleus after IKBKE knockdown.The second part explores the mechanism of IKBKE's effect on Snail14.Total proteins of glioma cell lines U87 and LN229 were extracted,and the expressions level of IKBKE and Snail1 were verified.Anti-IKBKE antibody was used to immunoprecipitate total proteins of the two cell lines.Then,WB was used to verify the compounds obtained by immunoprecipitation,and anti-snail1 antibody was used to identify whether there was Snail1 protein in the protein compounds obtained by anti-IKBKE binding.Then,the same method was used for reverse validation.Anti-snail1 was used for immunoprecipitation of the total protein extracted from the cells,WB was used for verification of the compounds obtained by immunoprecipitation,and anti-IKBKE antibody was used to identify whether there was IKBKE protein in the anti-snail1 binding protein.5.The expression levels of Snail1 in U87 and LN229 cells and U87-shIKBKE and LN229-shIKBKE cells were treated with proteasome inhibitor MG132(20uM)and were detected and compared by Western blot.CHX(50uM)was used to inhibit the synthesis of Snail1 protein,and the protein was harvested at 0,8,16,24 and 32 hours.Then western blot analysis was performed to detect dynamic changes of Snail1protein degradation after CHX treatment in U87?LN229 cells and U87-shIKBKE,LN229-shIKBKE cells.Result1.The results of scratch experiment showed that,compared with the control group,the migration distance,migration ability and scratch healing area of cells in the IKBKE-shRNA treatment group of U87 and LN229 cells were shortened,while the migration distance,migration ability and scratch healing area were increased in the IKBKE treatment group of U118 cells.The results showed that the downregulation of IKBKE expression significantly inhibited the migration ability of U87 and LN229 cells,and the upregulation of IKBKE expression significantly promoted the migration ability of U118 cells in the experimental group compared with the control group.Transwell basement membrane assay showed that the number of invaded cells in U87 and LN229 cells was significantly lower in the IKBKE-shRNA group than in the control group,and the number of invaded cells in U118 cells was significantly higher in the overexpressed IKBKE group than in the control group.These results suggested that the invasion ability was significantly decreased in U87 and LN229 cells with low IKBKE expression,and was significantly enhanced in U118 cells with overexpression of IKBKE.2.Western bloting showed that both IKBKE and Snail1 proteins were highly expressed in glioma cell lines.Moreover,the expression of IKBKE was positively correlated with the expression of Snail1 in glioma.3.Western bloting showed that in U87 and LN229 cells,after knockdown of IKBKE gene expression level,the level of total Snail1 protein was significantly decreased,while the level of E-cadherin protein was increased.After knockdown of IKBKE gene,the content of Snail1 protein in the cytoplasm was increased,while the content of Snail1 protein in the nucleus was decreased.Immunofluorescence results showed the same results.4.Western blot verified the expressions of IKBKE and Snail1 in U87 and LN229 glioma cells after extracting the total proteins of U87 and LN229 glioma cells,respectively.Anti-IKBKE was used for immunoprecipitation,and Western blot verified that the anti-IKBKE binding protein contained Snail1 protein.Reverse Western blot verified that the anti-snail1 binding protein contained IKBKE protein.5.Using proteasome inhibitor MG132(20uM)to treat U87 and LN229 cells and U87-shIKBKE and LN229-shIKBKE cells,Western blot results showed that whether MG132 was added or not,the expression of Snail1 protein in IKBKE knockdown group was significantly lower than that in control group.In addition,the expression of Snail1 protein in IKBKE knockdown group and control group increased significantly after MG132 was added.To evaluate the dynamic process of Snail1protein degradation,we used CHX(50uM)to inhibit its protein synthesis,harvested the protein at 0,8,16,24,and 32 hours,and then performed Western blot experiments.The degradation rate of Snail1 protein in glioma cells with low IKBKE was faster than that in U87 and LN229 cells.Conclusion1.By regulating and promoting the expression level of transcription factor Snail1,IKBKE inhibits the expression level of E-cadherin,thus leading to EMT and promoting the invasion and migration ability of glioma cells.On the contrary,silencing IKBKE can reduce the expression level of transcription factor Snail1 and restore the expression level of E-cadherin to normal,thereby inhibiting the invasion and migration of glioma cells.2.IKBKE expression and Snail1 expression in the tissue of gliomas were positively correlated.After knockdown of IKBKE gene expression level,the level of total Snail1 protein was significantly decreased,while the level of E-cadherin protein was increased,suggesting the existence of IKBKE-Snail1-E-cadherin pathway.After knockdown of IKBKE gene,the content of Snail1 protein in the cytoplasm increased,while the content of Snail1 protein in the nucleus decreased,suggesting that knockdown of IKBKE gene led to the transfer of Snail1 from the nucleus to the cytoplasm.3.This study found that IKBKE could directly bind the transcription factor Snail1 and stablilized Snail1.IKBKE regulated the protein level of Snail1 by inhibiting the degradation of Snail1.