IKBKE Promotes Glioblastoma Progression By Establishing The Regulatory Feedback Loop Of IKBKE/YAP1/miR-Let-7b/i

objectiveIn the latest study,IKBEK(inhibitor of nuclear factor kappa-B kinase subunit epsilon)was overexpressed in various types of malignant tumors.Our study has also confirmed that IKBKE is highly expressed in glioblastoma and is positively correlated with the grade of human glioblastoma.Silencing IKBKE protein expression can significantly inhibit the proliferation of human brain glioma cells.,invasion and migration capabilities.In this experiment,we silenced/overexpressed the expression of IKBKE,YAP1 and miR-Let-7b/i,to investigate the effect of IKBKE on the malignant progression of human brain glioblastoma through the IKBKE/YAP1/miR-Let-7b/i loop pathway.MethodsWe selected the human brain glioblastoma cell line U87-MG cell line and LN-229 cell line for this study.The IKBKE shRNA lentivirus was first constructed.The IKBKE shRNA lentivirus was used to transfect the U87-MG cell line and LN-229 cell line to downregulate the IKBKE protein expression in the cells.RT-PCR and Western blot methods were used to detect the expression of IKBKE mRNA and protein in the cells;CCK-8 method was used to compare the changes of the proliferation ability of the control group,NC group and shIKBKE group;Transwell method was used to detect the changes of migration and invasive ability of each group of cells;after the effect of silencing IKBKE gene was clearly determined,the total YAP1,total p-YAP1(S127),YAP1 in cytoplasm and YAP1 in cytoplasm were detected by Western blot.The changes of mRNA levels of YAP1 and miR-Let-7b/i after silencing IKBKE gene were detected by RT-PCR.Immunofluorescence assay was used to detect changes in total YAP1,cytoplasm,and nucleus YAP1 in the cells,after silencing IKBKE gene.The IKBEK overexpression plasmid was constructed and the IKBEK overexpression plasmid was used to transfect the cells.RT-PCR and Western blot were used to detect the over-expression level of IKBKE protein and mRNA,and the changes of YAP1,miR-Let-7b/i protein and mRNA levels.YAP1 siRNA was constructed and transfected into cells.YAP1 protein and mRNA expression levels were detected by Western blot and RT-PCR methods,the group with the best knockdown effect was selected for the next experiment.After clear knockdown effect of YAP1,the expression levels of IKBKE,miR-Let-7b,and miR-Let-7i were detected by Western blot and RT-PCR.A Flag-YAP1 overexpression plasmid was constructed and transfected to cells that silence the IKBKE gene.The level of YAP1 overexpression was detected by Western blot and RT-PCR,and the expression levels of IKBKE,miR-Let-7b and miR-Let-7i were detected.MiR-Let-7b mimics and miR-Let-7i mimics were constructed and transfected.The level of overexpression was detected by RT-PCR.The IKBKE,total YAP1,nuclear YAP1 and cytoplasmic YAP1 protein levels were detected by Western blot.Results1.Silencing of IKBKE gene expression: RT-PCR and Western blot methods showed that after transfection of IKBKE shRNA lentivirus,the protein level and mRNA level of IKBKE in shIKBKE group cells were significantly reduced;CCK-8 experiment results showed that after IKBKE gene silencing,the value-added ability of cells decreased significantly compared with the control group,and the value-added curve was significantly different.Transwell experiment results showed that in the Matrigel or Matrigel-free small rooms,the average number of cells transfected with IKBKE shRNA lentiviral cells through the polycarbonate membrane was significantly reduced;Western blot experiments showed that after silencing IKBKE gene,the total YAP1 and nucleus YAP1 protein levels in the cells were decreased,and the total p-YAP1(S127)and YAP1 protein levels in the cytoplasm were increased.Immunofluorescence experiments showed that after silencing the IKBKE gene,the total amount of YAP1 in the cells decreased,the YAP1 in the nucleus decreased,and YAP1 in the cytoplasm increased;RT-PCR results showed that after silencing the IKBKE gene,YAP1 mRNA levels did not change significantly,the levels of miR-Let-7b and miR-Let-7i were significantly increased.2.Overexpression of IKBKE gene: RT-PCR and Western blot analysis showed that after IKBKE overexpression plasmids were transfected into cell lines,IKBKE protein levels and mRNA levels were significantly higher than those of control and NC group cells;Western blot experiments showed that after overexpression of IKBKE gene,the total YAP1 protein level was increased and the total p-YAP1(S127)protein level was decreased.RT-PCR results showed that there was no significant change in YAP1 mRNA level after overexpression of IKBKE gene,miR-The levels of Let-7b and miR-Let-7i decreased significantly.3.Silencing YAP1 gene expression: RT-PCR and Western blot methods showed that after transfection of YAP1 siRNA,the YAP1 protein level and mRNA level of the YAP1 siRNA group were significantly lower than those of the control and NC group.RT-PCR results showed that the level of miR-Let-7b and miR-Let-7i was significantly increased after silencing YAP1 gene.4.Overexpression of YAP1 gene: RT-PCR and Western blot analysis showed that after transfection of YAP1 overexpression plasmid with silencing IKBKE cell line,compared with the control group and silenced IKBKE group cells,the protein level and mRNA level of YAP1 transfected with YAP1 plasmid group were significantly increased.RT-PCR results showed that after overexpression of YAP1 gene,miR-Let-7b and miR-Let-7i levels were significantly reduced.5.Over-expression of miR-Let-7b/i: RT-PCR showed that after transfection of miR-Let-7b/i mimics,compared with control and NC cells,the expression level of miR-Let-7b/i in transfected miR-Let-7b/i mimics group was significantly increased.Western blot experiments showed that after overexpression of miR-Let-7b/i,the total YAP1,total IKBKE,and nucleus YAP1 protein levels were decreased,and the YAP1 protein level in cytoplasm was increased.Conclusion1.Silencing IKBKE gene can significantly inhibit the proliferation of human glioma cells.2.Silencing IKBKE gene can significantly inhibit the migration and invasion of human glioma cells.3.Silencing IKBKE gene can reduce the total YAP1 and nuclear YAP1 protein levels,increase the total p-YAP1(S127),cytoplasmic YAP1 protein levels,but no significant changes in mRNA levels.4.Silencing IKBKE gene can increase the expression of miR-Let-7b and miR-Let-7i.5.Overexpression of IKBKE gene can increase the total YAP1 protein level and decrease the total p-YAP1(S127)protein level;however,there is no significant change in mRNA level.6.Overexpression of IKBKE gene reduced the expression of miR-Let-7b and miR-Let-7i.7.Silencing YAP1 gene can increase the expression of miR-Let-7b and miR-Let-7i.8.Overexpression of YAP1 gene reduced the expression of miR-Let-7b and miR-Let-7i.9.Overexpression of miR-Let-7b/i,the total YAP1,total IKBKE and YAP1 protein levels in the nucleus can be decreased,and the YAP1 protein level in the cytoplasm is increased.In summary,IKBKE can regulate the malignant progression of human brain glioblastoma through the IKBKE??YAP1??miR-Let-7b/i??IKBKE? loop pathway.