Molecular Mechanism Of Suppressing Epithelial-Mesenchymal Transition In Non-Small-Cell Lung Cancer Based On The Mediation Of Transcription Factor FOXM1

Objective:Lung cancer is one of the most malignant cancer with the highest morbidity and mortality worldwide,and around 80% of which is Non-small-cell lung cancer(NSCLC).NSCLC patients with advanced stage often develop systematic metastasis and the five-year survival rate is extremely low.Epithelial-mesenchymal transition(EMT)plays an essential role in cancer progression,and has been widely recognized as the original source of invasion and metastasis in NSCLC.In EMT initiation,plenty of transcription factors gathered into nucleus,thereby enhancing the expression of EMT-related proteins.Substantial evidence shows that Forkhead box(FOX)gene family plays a critical role in regulating EMT program,and in which,FOXM1 can modulate multiple malignant process in many type of tumor cells.However,the modulate functions of FOXM1 on EMT in NSCLC cells,especially the transcriptional function on E-cadherin coding gene CDH1,remain unclear.This work mainly focused on FOXM1 and its mechanism in regulating EMT in NSCLC cells,and we also ecaluated FOXM1 inhibitor thiostrepton’s effects in EMT intervention.Method:(1)We altered protein level of FOXM1 to investigate its fuction in regulating EMT in NSCLC cells: Using HE stain to observe morphology change of NSCLC cells after overexpression of FOXM1;Using scratch assay and transwell assay to detect the alterration of migration and invasion ability of NSCLC cells after overexpression or knockdown of FOXM1 respectively;The expression of EMT biomarkers after overexpression or knockdown of FOXM1 were analyzed by western-blot.(2)Molecular mechanism discussed the transcriptional regulation of FOXM1 on EMT biomarkers: Using Dual-Luciferase report assay to verify the CDH1 promoter regulation by FOXM1;Using Chromatin Immunoprecipitation(Ch IP)to confirm the CDH1 promoter binding role of FOXM1;Knockdown Snail in FOXM1-overexpressed cells,and reveal the overall transcriptional regulation of FOXM1 on EMT biomarkers by Dual-Luciferase report assay and Immunofluorescence.(3)Anti-EMT effect of FOXM1 inhibitor thiostrepton on NSCLC cells in vitro: NSCLC cells’ viability after thiostrepton treatment were measured by MTT assay;Colony formation assay was used to determine the effect of thiostrepton on colony formation ability of NSCLC cells;The reversion of thiostrepton on TGF-β1-induced EMT and mobility were analyzed by morphological observation,Scratch assay,transwell and Western-Blot.Resuts:(1)GSEA analysis and the detection of expression level of EMT markers and FOXM1 in NSCLC cells indicate that FOXM1 is relevant to EMT;After overexpression of FOXM1,cells demonstrated epithelial-mesenchymal intermediate phenotype,rather than entire mesenchymal phenotype;Migration and invasion ability were enhanced in FOXM1-overexpressed A549 cells;protein expression of N-cadherin,Snail,Vimentin were increased while E-cadherin was decreased.Downregulate FOXM1 in H1975 cells can inhibit cell migraion and invasion,and protein level of E-cadherin was increased,whereas N-cadherin,Snail,Vimentin were decreased.(2)Dual-Luciferase Assay showed that FOXM1 can inhibit CDH1-2000-promoter luciferase activity;Ch IP assay confirmed that FOXM1 can endogenously bind to CDH1 promoter;After downregulation of Snail in overexpressed-FOXM1 cells can rescue the expression of E-cadherin.(3)MTT showed thiostrepton can inhibit proliferation of NSCLC cells;Clone formation assay displayed that thiostrepton can abolish cloning formation ability of NSCLC cells;TGF-β1-induced EMT process was accompanied with increased level of FOXM1,which,however,can be reversed by thiostrepton through morphology observation;Cell scratch assay and transwell assay revealed that thiostrepton can suppress TGF-β1-induced mobility and invasiveness;Western-Blot tested that thiostrepton can downregulate TGF-β1-induced growth level of FOXM1,N-cadherin,Snail,Vimentin,and block TGF-β1-induced loss of E-cadherin.Conclusion: Overexpression of FOXM1 can accelerate EMT progression in NSCLC cells;Downregulate FOXM1,however,can refrain this process.FOXM1 can inhibit transcription of E-cadherin,and it can provoke Snail expression to suppress E-cadherin expression indrectly,therefore controling EMT program as a whole.FOXM1 inhibitor thiostrepton displayed as a suppressor in blockoing NSCLC cells’ proliferation,colony formation and EMT.All the experiments above indicate that changing expression of FOXM1 can inhibit EMT process.