Mechanism Study On Expression Of IKKα/IKKβ/NFκB Regulated By E2F1in Esophageal Cancer Cell Lines ECA109、KYSE150

Objective: SURVIVIN is found that the strongest apoptosis inhibiting factor,promotes cell proliferation, inhibition of apoptosis, the molecular mechanism ofcontinuous high expression in tumor tissue had not been reported. Transcription factorE2F1also plays a very important role in promoting cell proliferation. IKK/IKKβ/NFκBsignaling pathway is an important part in regulating the expression of SURVIVIN. Inesophageal squamous cell carcinoma cell lines, whether E2F1involved inIKK/IKKβ/NFκB expression regulation of signaling pathways, whether through thesignaling pathways involved in the expression of SURVIVIN further, there is no researchdata.Methods: This study intends to research in esophageal squamous cells, E2F1inhibitors inhibit the expression of the E2F1mRNA and protein, the establishment ofE2F1over-expression and shRNA plasmid vector, plasmid electrical transfection ofesophageal squamous cell carcinoma cell lines, qPCR and Western blot to know therelationship between E2F1with IKK/IKKβ/NFκB/SURVIVIN signaling pathways intranscription level and protein level. Flow cytometry to detect the cell cycle and cellapoptosis, to know E2F1affecting the biological functions of esophageal squamouscancer cells. ChIP experiment shown binding sites of E2F1protein in IKK/IKKβ/NFκBgene promoter region. Double fluorescein report methods further proved E2F1proteinbinding the IKK/IKKβ/NFκB gene promoter region.Results: In the esophageal cancer cell lines ECA109and KYSE150, usedE2F1inhibitors BZB to induct48hours and24hours, E2F1was inhibited intranscriptionlevel and protein levels at the same time, accompanied by the down- regulated of IKKβ/NFκB/SURVIVIN in transcription level and protein level.InECA109cell lines, IKK expressed higher in transcription level but lower in protein level.InKYSE150cell lines, IKK expressed lower in transcription level and proteinlevel.Establishmentof E2F1over-expression and shRNA plasmids carrier, after E2F1over-expression plasmid vectors electric transfected in the two cell lines, E2F1expression increased in transcription level and protein level, accompanied byup-regulated expression of IKKβ, NFκB, SURVIVIN in transcription level and proteinlevel.In ECA109cell lines, IKK decreased in transcription level and protein level; InKYSE150cell lines, IKK had no obvious change in transcription level butdown-regulated expression in prot-ein level.ShRNA plasmid vector electric transfectionin the two cell lines, E2F1decreased in transcription level and protein level,accompanied by the down-regulated expression of IKKβ, NFκB, SURVIVIN intranscription and protein level: In ECA109cell lines, IKK increased in transcription andprotein level.In KYSE150cell lines, E2F1decreased in transcription and proteinlevel.Cell cycle and cell apoptosis dete-ction, E2F1promoted G1/S phase transformationin the two cell lines, played the role of promoting cell proliferation, S phase cellspercentage increased obviously. when the E2F1gene was silenced by E2F1shRNA, thefunction of the cell proliferation was significantly inhibited, S phase cells percentagedecreased, the difference was statistically significant (P＜0.05). E2F1inhibited cellapoptosis in the two cell lines, when E2F1was silenced by E2F1shRNA, early apoptosiscells increased significantly, the difference was statistically significant (P＜0.05).Chromatin immunecoprecipitation experiments shown E2F1protein had binding site inIKKβ gene promoter region in ECA109cell lines, we have not found the binding sites inIKK, IKKβ, NFκB gene promoter region of E2F1protein in KYSE150cell lines.Double fluorescein enzyme report gene experiments confirmed that the group of totaltransfection pGV142-E2F1, pGL3-IKKβ, pRL-TK, fluorescein enzyme activity of thisgroup was obv-iously enhanced, the difference was statistically significant (P＜0.05),and once aga-in proved the E2F1protein binded with IKKβ gene promoter region.Conclusion: In ECA109and KYSE150esophageal squamous cancer cell lines,there were regulation relationship between E2F1and SURVIVIN, E2F1participated inSURVIVIN regulation mechanism, the molecular mechanisms may be through theIKKβ/NFκB classic signal pathways.In ECA109cell lines, E2F1protein was likely tocombine IKK β promoter region within-751bp to participate in the regulation of NFκB, and than the expression of SURVIVIN. This study found the continuous high expressionmolecular mechanisms of SURVIVIN in cancerous tissue for the first time, the researchprobably established a new regulation pathway, the discovery of this new regulation willprovide new targets for clinical drug intervention.