Transcription Factor Sp1 And E2F1 Regulate AQP3 Expression To Affect Cell Migration And Invasion

I.ObjectiveAquaporins(AQPs)are small,intrinsic membrane proteins that facilitate the transport of water,glycerin and urea across cell plasma membranes and are involved in numerous physiological as well as pathophysiological processes.AQP3 is highly expressed in the endometrium of the implantation window and can promote the migration and invasion of endometrial cancer cells and promote the smooth progress of embryo implantation.However,there are few studies on the regulation of AQP3 transcription.Sp1,as the first transcription factor found in mammalian cells,mediated tumor growth and metastasis by regulating gene transcription.The E2 F family is mainly involved in cell cycle and apoptosis.E2F1 is a transcription factor,which involves cell cycle,cell proliferation,apoptosis and differentiation.This paper intends to predict the transcription factors associated with AQP3 promoter,analyze whether the transcription factor Sp1 and E2F1 are binded to AQP3 promoter,activate the transcription of AQP3;explores Sp1,E2F1 transcription factors regulate AQP3 expression and affects human endometrial cancer cell migration and invasion ability of the molecular mechanisms.II.Methods1.Through the bioinformatics method,the transcription factors combined with AQP3 promoter were predicted,and the transcription factors Sp1,E2F1 and NF-kappa B1 were used as the initial research objects.2.The si RNA(si-NC,si-Sp1,si-E2F1 and si-NF-kappa B1)were transfected into Hela cells using liposome transfection method,and the transcription factors that activated AQP3 transcription were preliminarily screened by real-time PCR.3.The NC+Basic(si-NC+pGL3-basic+pRL-TK)、 NC+AQP3-promoter(si-NC+pGL3-AQP3-promoter +p RL-TK)、 si-Sp1+AQP3-promoter(si-Sp1+p GL3-AQP3-promoter+p RL-TK)、 AQP3-promsi-E2F1+oter(si-E2F1+p GL3-AQP3-promoter+p RL-TK)、si-Sp1+Basic(si-Sp1+p GL3-basic+p RL-TK)、 si-E2F1+Basic(si-E2F1+p GL3-basic+p RL-TK)were transfected into Hela cells using liposome transfection method,through luciferase report assay,it was analyzed whether the transcription factor Sp1 and E2F1 were combined with AQP3 promoter.4.The binding site of Sp1 on AQP3 promoter was predicted by bioinformatics method,and S1(-615/+22)was constructed on the p GL3-basic carrier.5.The NC+Basic(si-NC+p GL3-basic+p RL-TK)、 si-Sp1+S1(si-Sp1+p GL3-AQP3-S1(-615/+22)+ p RL-TK)、Empty vector+S1(Empty vector+p GL3-AQP3-S1(-615/+22)+p RL-TK)、Sp1 vector +S1(Sp1 vector + p GL3-AQP3-S1(-615/+22)+p RL-TK)were transfected into Hela cells using liposome transfection method,through luciferase report assay,it was analyzed where transcription factor Sp1 specific binds to on the AQP3 promoter.6.In Hela cells,the specific binding of the transcription factor Sp1 to AQP3 promoter was verified by chromatin immunoprecipitation experiment and PCR experiment.7.In human endometrial cancer cell Ishikawa and HEC-1A cells,by Western blot,real-time PCR and immunofluorescence assay,the expression and distribution of transcription factor Sp1 and E2F1 were detected.8.Through liposome transfection,Sp1 overexpressed plasmid,si-Sp1 and si-E2F1 were transfected into human endometrial cancer cell line Ishikawa and HEC-1A cells.9.In human endometrial cancer cells,Ishikawa and HEC-1A cells were tested by Western blot to detect the effect of different concentrations of Mith A on the expression of Sp1 protein.10.In human endometrial cancer cells,Ishikawa and HEC-1A cells were tested by Western blot and real-time PCR to detect the effect of transcription factor Sp1 on AQP3 expression.11.In human endometrial cancer cells,Ishikawa and HEC-1A cells,Transwell and scratch-wound assay were used to detect the effects of transcription factor Sp1 on the migration and invasion ability.12.In human endometrial cancer cell line HEC-1A cell,the effect of transcription factor E2F1 on NF-kappa B signaling pathway and AQP3 expression was detected by Western blot and real-time PCR.III.Results1.The transcription factor Sp1 binds to AQP3 promoter to activate the transcription of AQP3,while E2F1 and NF-kappa B1 cannot be combined with AQP3 promoter to activate the transcription of AQP3.2.The expression of transcription factor Sp1 and E2F1 in endometrial cancer cell HEC-1A cells was higher than that of Ishikawa,and the expression position was in the nucleus.3.The expression of Sp1 was decreased with the increase of Mith A concentration in human endometrial cancer cells Ishikawa and HEC-1A cells.4.In human endometrial cancer cells,Ishikawa and HEC-1A cells,the Sp1 was down-regulated to reduce the expression of AQP3,and the cell migration and invasion ability were reduced.5.In human endometrial cancer cells,Ishikawa and HEC-1A cells,the Sp1 was up-regulated and the expression of AQP3 was increased to promote cell migration and invasion.6.In the endometrial cancer cell HEC-1A,E2F1 was down-regulated to activate the NF-kappa B signaling pathway and reduce the expression of AQP3.IV.Conclusions1.The transcription factor Sp1 activates AQP3 transcription by specific binding to AQP3 promoter.2.In human endometrial cancer cells,Ishikawa and HEC-1A cells,Sp1 promotes cell migration and invasion by promoting the expression of AQP3.3.In the endometrial cancer cell HEC-1A,E2F1 increases the expression of AQP3 by reducing the activation of NF-kappa B signaling pathway.