The Role And Proliferative Mechanism Of FOXM1 In Esophageal Squamous Cell Carcinoma

Objective:Esophageal carcinoma is a common malignancy of the upper digestive tract,with over 570,000 new cases globally in 2018.Esophageal squamous cell carcinoma(ESCC)represents the major histological type of esophageal carcinoma,accounting for about 84%of all cases.Due to the limitation of clinical methods for early diagnosis and treatment,ESCC still has a poor 5-year survival rate.Thus,it is urgent to explore the pathogenesis mechanisms of ESCC and provide new insights into the early diagnosis and clinical treatment of ESCC.FOXM1 is overexpressed in a variety of solid human tumors,and participates in many tumor biological processes including cell proliferation,cell invasion and migration,DNA damage repair.Therefore,FOXM1 has been widely recognized as a potential key target for human cancer therapies.However,the significance of FOXM1 in ESCC remains largely unknown.This study aims to explore the potential biological function and target gene network of FOXM1 in ESCC,and further study the proliferation mechanism of FOXM1,which will help to develop more effective therapeutic strategies for ESCC.Methods:1.The biological function and predicted target gene network of FOXM1 in ESCCFirstly,the pan cancer expression of FOXM1 was analyzed,and the relationship between FOXM1 expression and immune checkpoint genes,DNA repair genes and methyltransferases in Pan cancer datasets were preliminarily explored;Secondly,based on GSEA algorithm,the biological function of FOXM1 in ESCC was studied;Then,the FOXM1 predicted target gene network was constructed based on the MEGANA coexpression network and transcriptional binding site analysis;Finally,it was found that CDC6 was a key target gene of FOXM1 through PPI and correlation analysis.2.Expression of FOXM1 and CDC6 in ESCCSix public datasets of ESCC were analyzed to preliminarily evaluate the mRNA expression level of FOXM1 and CDC6 in ESCC.We further detected FOXM1 and CDC6 expression in both adjacent normal esophageal squamous epithelium and ESCC tissues by immunohistochemistry.Finally,we detected the mRNA and protein expression levels of FOXM1 and CDC6 in ESCC cell lines and normal cell lines(HET-1 A)by qRT-PCR and western blot,respectively.3.Explore the molecular mechanism of FOXM1 regulating CDC6After gradient transfected ESCC cell with FOXM1 overexpression vector or siRNA respectively,the mRNA and protein expression levels of CDC6 were detected.The potential FOXM1 binding sites in the promoter region of CDC6 were analyzed and predicted by online database.The binding sites and transcriptional activity of FOXM1 in the promoter region of CDC6 were verified by chromatin immunoprecipitation assay and dual-luciferase reporter assay to determine whether CDC6 is the direct target gene of FOXM1.4.Investigate the biological functions of FOXM1 and CDC6 in malignant proliferation of ESCCThe effects of FOXM1 and CDC6 on proliferative capacity and clonogenic ability of ESCC were evaluated using CCK-8,EdU,colony formation assays.We carried out an interference and rescue experiment to confirm whether FOXM1 promotes ESCC proliferation by promoting CDC6 expression.The effects of FOXM1 and CDC6 on cell cycle progression in ESCC cells were explored by flow cytometry.Results:1.The biological function and predicted target gene network of FOXM1 in ESCCFOXM1 was significantly up-regulated in 33 tumors compared with the normal control group,and was associated with immune checkpoint genes,DNA repair genes and methyltransferases;The results showed that the main biological functions of FOXM1 involved in cancer-related pathways,DNA replication and cell cycle regulation,DNA damage response and repair;The FOXM1 predicted target gene network in the background of ESCC was constructed by MEGANA analysis;CDC6 was screened as a new key target gene of FOXM1 in ESCC.2.Over expression of FOXM1 and CDC6 in ESCC tissues and cell linesFOXM1 and CDC6 mRNA expressions were significantly up-regulated compared with adjacent tissues in the six ESCC datasets.The positive expression levels of FOXM1 and CDC6 in ESCC tissues were significantly higher than that in adjacent non-cancerous tissues by IHC analysis.Both mRNA and protein levels of FOXM1 and CDC6 were significantly upregulated in ESCC cell lines compared to HET-1A cells,respectively.3.FOXM1 regulates CDC6 expression through binding to the CDC6 promoterExperiments of gradient overexpression or interference with FOXM1 suggested FOXM1 positively regulates CDC6 expression.Bioinformatics analysis results showed that the CDC6 promoter region contained four putative FOXM1-binding sites.We found that FOXM1 is bound to the DNA fragment from two binding sites in the CDC6 promoter region by ChIP-PCR assay.Luciferase reporter assay showed that FOXM1 could activate the expression of CDC6 by binding to the BS2 sequence of the CDC6 promoter.4.FOXM1 promotes ESCC cell proliferation depending on the positive regulation of CDC6 expressionAfter overexpression of FOXM1 or CDC6,cell proliferation rate and clonogenic ability of ESCC cells were significantly enhanced.Futhermore,after forced overexpression of CDC6 in FOXM1 knockdown ESCC cells,the cell proliferation rate and colony forming ability suppressed by FOXM1 knockdown could be partially eliminated by CDC6 overexpression.After overexpression of FOXM1 or CDC6,the proportion of S-phase cells increased significantly and G1-phase cells decreased significantly.Moreover,overexpression of CDC6 could partially reverse the G1/S cell cycle arrest caused by FOXM1 knockdown.Conclusion:1.Bioinformatics analysis showed that FOXM1 may be mainly involved in biological processes such as cancer related pathways,DNA replication and cell cycle regulation,DNA damage response and repair in ESCC.2.Construct the predictive target gene network of FOXM1 in the context of ESCC,which is helpful to the functional analysis and verification of FOXM1.3.FOXM1 and CDC6 were abnormally highly expressed in ESCC tissues and cells,and showed a positive correlation.4.FOXM1 activates CDC6 expression by binding to the CDC6 promoter region.5.FOXM1 mediated proliferation of ESCC cells partly depending on the expression of CDC6,which may be related to promoting G1/S cell cycle transition.