FOXO1 Inhibits Stemness And Metastasis Of Nasopharyngeal Carcinoma By Antagonizing Its Binding Protein MYH9

BackgroundsNasopharyngeal carcinoma(NPC)is a special malignant head and neck tumor that occurs in South China.In clinical medicine,the main cause of death in patients with NPC is closely related to tumor cell metastasis and radio-chemotherapy resistance.FOXO1 is a classic tumor suppressor gene,however,its mechanism of regulating tumor metastasis in NPC has not been studied deeply,and it has not been reported in terms of suppressing tumor stemness.Contents and methods1.To explore the tumor stemness,metastasis and chemosensitivity functions of FOXO1 in NPC and the molecular mechanism(1).Construction of NPC cells stably transfected with FOXO1;Realtime-PCR(qPCR)and western blot analysis was used to determine the overexpression or interference efficiency;(2).The effect of FOXO1 on stemness,migration and invasion and chemoresistance of NPC cells was analyzed by sphere formation assay,flow cytometry analysis,migration,invasion and wound healing assay,MTT assay,and tumor xenograft study in vitro and in vivo.(3).Western blot analysis was adopted to determine the expression of β-catenin,P53,TCF4,ZEB1,c-Myc,OCT4,Vimentin,E-Ca,N-Ca in NPC cells.2.To identify the proteins interacting with FOXO1.(1).Coimmunoprecipitation(Co-IP)assay combined with mass spectrometry-based quantitative proteomics was adopted to identify the interactive proteins of FOXO1.(2).Co-IP assay combined with western blot analysis to verify the proteins that interact with FOXO1 endogenously and exogenously;(3).The colocalization of the interactive protein and FOXO1 in NPC cells were detected by immunofluorescence and confocal microscopy;(4).qPCR and Western blot analysis were applied to investigate the relationship between FOXO1 and the interacting protein in NPC cells;3.To explore miRNA that directly target MYH9(1).Bioinformatics analysis was used to predict miRNAs targeting MYH9 3’UTR;(2).qPCR and Western blot analysis were used to investigate the relationship between the miRNA and MYH9 in NPC cells;(3).Luciferase reporter assay was performed to verify that the miRNA targets MYH9;(4).Several in vitro functional experiments were performed to observe the role of miRNA when FOXO1 inhibits tumor stemness,metastatic invasion,and promotes chemotherapy sensitivity in NPC;(5).Western blot experiment was used to detect the role of miRNA when FOXO1 regulates downstream factor protein levels;4.To explore the transcription factor which binds the promoter region of miR-133a-3p(1).Bioinformatics analysis was used to predict transcription factor that regulates miR-133a-3p;(2).qPCR analysis was performed to verify the regulation of transcription factors on miR-133a-3p;(3).Chromatin immunoprecipitation(Ch-IP)assay was applied to detect the binding of transcription factors to the miR-133a-3p promoter region;(4).Using electrophoretic mobility shift assay(EMSA)to validate the binding of the transcription factor to miR-133a-3p promoter region;(5).Luciferase reporter assay was performed to verify the acceleration of miR-133a-3p transcription by the transcription factor.(6).Western blot was used to detect the expression of P53 downstream related proteins;(7).Several in vitro functional experiments were performed to observe the role of P53 when FOXO1 inhibits tumor stemness,metastatic invasion,and promotes chemotherapy sensitivity in NPC;(8).Western blot experiment was used to detect the role of P53 when FOXO1 regulates downstream factor protein levels;5.Analysis of the expression characteristics and correlation of FOXO1 and MYH9 in NPC(1).To investigate the clinical expression characteristics of FOXO1 by QPCR analysis,immunohistochemistry(IHC)assay and related statistical analysis;(2).To investigate the clinical expression characteristics of MYH9 by QPCR analysis,immunohistochemistry(IHC)assay and related statistical analysis;(3).Correlation analysis between FOXO1 and MYH9 expression;(4).Univariate and multivariate analysis were used to explore the independent factors of NPC patients.Results(1).FOXO1 inhibits tumor stemness,metastasis and promotes sensitivity to chemotherapy of NPC in vivo and in vitro;(2).FOXO1 interacts with MYH9 and inhibits MYH9 expression at the post-transcription level;(3).miR-133a-3p directly targets MYH9;(4).P53 binds the promoter region of miR-133a-3p and induces its transcription;(5).FOXO1 is relatively low-expressed in NPC tissues and is positively correlated with patient prognosis;MYH9 is relatively high-expressed in NPC tissues and negatively correlated with patient prognosis;FOXO1 and MYH9 are negatively correlated;FOXO1 is an independent prognostic factor in NPC.ConclusionsFOXO1 inhibits the expression of its interacting protein MYH9 by regulating the P53/miR-133a-3p signal axis,thereby inhibiting the β-catenin/TCF4/ZEB1,which is the downstream pathway of MYH9,and ultimately inhibit the tumor stemness and metastasis and promote chemotherapy sensitivity in NPC.