FOXML1 Transcription Factor Promotes Hepatocellular Carcinoma Progression By Regulating The Expression Of KIF4A

Background: Liver cancer is the fourth leading cause of cancer-related death worldwide and Hepatocellular carcinoma (HCC) represents the most common type of liver cancer,accounting for approximately 95%.As the strong ability of proliferation,invasion and metastasis of HCC cells and paitients with HCC are insensitivity to radiotherapy or chemotherapy,surgical resection is still the main treatment method.However,the majoraty of the patients with HCC were diagnosed in the middle or late stages and only 10-23% of the patients were suitable for surgical treatment.Thus it’s a really critical task to find ways for early diagnosis and improvement of the sensitivity for radiotherapy or chemotherapy for HCC patients.Although a growing number of related researches were performed in order to explain the tumorigenesis process of HCC,the molecular basis still remains obscure.Therefore,revealing the complicated molecular mechanism of pathogenesis and development of HCC is currently an urgent requirement.FOXM1 is a proliferation associated conserved transcriptional modulator belonging to the forkhead box superfamily proteins and plays a critical role in cell cycle progression,specifically in the regulation of G1/S and G2/M transitions.FOXM1 is barely expressed in quiescent cells while its expression is significantly increased during cell proliferation,which controls cell DNA replication and mitosis by regulating the transcription of genes related to cell cycle.FOXM1 plays an important role in DNA damage repair,angiogenesis,tissue regeneration,apoptosis,invasion and migration of tumor cells and chemotherapy resistance.Therefore,exploring the role of FOXM1 and the underline mechanisms in the process of tumorigenesis and development will help us clarify the pathogenesis of tumors and provide theoretical basis for the discovery of new therapeutic targets for cancer.It is believed that the malignant proliferation of HCC is caused by a series changes of genes and their regulatory networks.As is known to all,the transcription factor FOXM1 and its regulating networks of downstream target genes play a pivotal role in HCC progression.However,the exact mechanism of tumorigenesis is still not clarified.What are the genes and regulatory networks participating in HCC progression? How are FOXM1 and its regulating targets activated in HCC? How do FOXM1 and its regulatory targets function in HCC tumorigenesis and progression? To address these questions,we choose HCC as the research subject and focus on FOXM1 and its regulatory networks,and to carry out further studies from bioinformatics,clinical cohort,molecular biology and cellular function in order to find new theoretical basis for gene diagnosis and molecular targeted therapy of HCC.Materials and methods:1.Bioinformatics analysis for differentially expressed gene in HCC Firstly,we conducted meta analysis based on the HCC microarray dataset of the expression profile from the public data platform of GEO database,trying to find significantly differentially expressed genes in HCC.Secondly,other differentially expressed genes were obtained from the clinical sample data of HCC from TCGA database by using bayesian analysis.Then,we performed cluster analysis according to these two groups of differentially expressed genes identified from GEO and TCGA database.Finally,we collected the shared genes from the cluster analysis and genes from the potential proliferation-associated genes to extract the up-regulated genes in HCC.2.Verificate the genes identified from bioinformatics analysis through clinical cohort study(1)HCC tissue samples were collected from clinical patients who received a surgery;the protein expressions of FOXM1 and KIF4 A in HCC tumor tissues and corresponding adjacent normal liver tissues were analyzed by western blot and immunohistochemical staining.(2)The "German semi-quantitative system" were used for statistical evaluation of immunohistochemical detection results.The Chi-square test were used for analyzing the association of FOXM1 and KIF4 A expression levels with different clinicopathologic characteristics in HCC.The Kaplan-Meier method and the log-rank test were used for the survival analysis of HCC patients.3.Explore the molecular mechanism of abnormal activation of FOXM1 and KIF4 A in HCC(1)We tested both of the protein and m RNA expression levels of KIF4 A upon FOXM1 overexpression or knockdown in HCC cells to determine the regulation of KIF4 A by FOXM1.(2)Bioinformatic analysis was performed to predict the potential upstream transcription regulators in KIF4 A regulation.The potential binding sites of FOXM1 transcription factor were identified on the promoter region of KIF4 A gene.(3)Chromatin immunoprecipitation(Ch IP)and Dual luciferase reporter assay were appllied to confirm whether KIF4 A is a direct target gene of FOXM1.4.Investigate the biological roles of FOXM1 and KIF4 A in the progression of HCC(1)HCC cell lines whicn FOXM1 or KIF4 A stabely overexpressed or silienced were generated by using the lenti-viral infection system;(2)The roles of FOXM1 or KIF4 A in the cell proliferation,colony formation,cell growth rate,cell viability and cell cycle progression of HCC cells were explored by Ed U assay,colony formation assay,cell growth curve,CCK-8 assay and cell cycle analysis.5.Explore the functions of FOXM1-KIF4 A axis in the malignant biological behavior of HCC(1)The HCC cell lines stablely overexpression of FOXM1 and siliencing of KIF4A (FOXM1 + sh KIF4A) were generated by applying the lenti-viral infection system.(2)The effects of FOXM1-KIF4 A axis on the proliferation of HCC cells were investigated by Ed U assay,colony formation assay,cell growth curve,CCK-8 assay and cell cycle analysis.(3)The roles of FOXM1-KIF4 A axis in HCC cell proliferation were varified by using the HCC xenograft model in vivo.Results: 1.Eighteen up-regulated genes were identified in HCC through bioinformatic analysis We identified 59 up-regulated genes and 51 down-regulated genes by using bioinformatic analysis of the data of HCC samples from GEO and TCGA databases according to the inclusion criteria.Then we performed cluster analysis between these 59 up-regulated genes and genes involved in HCC cell proliferation.We finally identified 18 up-regulated genes including FOXM1 and KIF4 A,which may participate in HCC cell proliferation.2.Both FOXM1 and KIF4 A were aberrantly elevated in human HCC tumor tissues and predicted a poor clinical outcome(1)Compared to the adjacent normal liver tissues,the protein levels of both FOXM1 and KIF4 A were highgly expressed in HCC tumor tissues by testing with western blot and immunohistochemistry.(2)The expression of FOXM1 and KIF4 A were positively correlated in HCC tumor tissues.(3)Correlation analysis of clinicopatholoic characteristics in HCC patients showed that the expression of FOXM1 was associated with pathological differentiation,tumor size,and TNM stages,and the expression of KIF4 A was associated with tumor size,pathological differentiation,vascular invasion and TNM stages.Furthermore,highly expression of FOXM1 or KIF4 A was associated with poor clinical outcomes.3.KIF4 A is a direct downstream target gene of FOXM1(1)Both of the protein and m RNA expression levels of KIF4 A were significantly up-regulated after gradient overexpression of FOXM1 in Hep G2,Sk-hep1 and Huh7 cells,whose endogenous level of FOXM1 were relatively low.(2)Both of the protein and m RNA expression levels of KIF4 A were significantly down-regulated after gradient knockdown of FOXM1 in Hep3 B and Huh7 cells whose endogenous level of FOXM1 were relatively high.(3)The bioinformatic predictive analysis through the UCSC online website showed that FOXM1 may be a potential upstream transcriptional regulator of KIF4A(4)Four potential binding sites (BS) of FOXM1 on the promoter of KIF4 A gene were identified by using bioinformatic analysis,and three of these four binding sites were confirmed through Ch IP assay.(5)The results of the dual luciferase reporter assay showed that overexpression of FOXM1 increased the luciferase activity of KIF4 A promoter,and mutation of binding site 3 (BS3: 5’-AGATGGAGT-3’) significantly abolished the luciferase activity induced by FOXM1,indicating that BS3 is the critical functional binding site for the interaction of FOXM1 to the promoter of KIF4 A.4.Both FOXM1 and KIF4 A promote the proliferation of HCC cells(1)Compared with the control group cells,the proliferation rate,the ability of colony formation,cell growth rate and cell viability of HCC cells stably expressing of FOXM1 or KIF4 A were significantly enhanced.(2)The percentage of Ed U positive cells in HCC cells stably knockdown of FOXM1 or KIF4 A was significantly decreased,and the ability of colony formation was reduced,the cell growth rate was slowed down,and the cell viability was also significantly weakened.(3)Compared with control HCC cells expressing a scrambled control sh RNA (sh-Control),those expressing sh RNA against FOXM1 (sh-FOXM1) were arrested in G1 phase along with less cells in S phase.(4)Knockdown the expression of KIF4 A,HCC cells could not accomplish the progression of mitosis and cytokinesis,resulting in the accumulation of cells in G2/M phase and multinucleate cells (> 4N cells).5.FOXM1 promotes HCC cell proliferation depending on KIF4A(1)The results of in vitro experiments showed that the cell proliferation rate,colony formation number,cell growth rate and cell viability were enhanced in FOXM1 + sh-Control cells relative to the Empty vector + sh-Control Hep G2 cells,but these effects were abrogated by KIF4 A knockdown (FOXM1 + sh-KIF4A).(2)Cell cycle analysis showed that HCC cells in the FOXM1 + sh-control group had a higher proportion of cells distributed in S phase compared with the Vector + sh-control group.However,this promotion effect of FOXM1 overexpression in Hep G2 cells was significantly blocked after interfering the expression of KIF4 A,as evidenced by a lower fraction of cells in S phase,G2/M phase arrest,and accumulation of multinucleate cells in FOXM1 + sh-KIF4 A group compared with the FOXM1 + sh-control group.(3)In vivo studies using a HCC mouse tumor xenograft model showed that compared to the control group,tumors in the FOXM1 + sh-Control group developed much more rapidly,but this effect was abrogated by KIF4 A knockdown (FOXM1 + sh-KIF4 A group).Tumors were smaller in FOXM1 + sh-KIF4 A than in FOXM1 + sh-Control mice,and FOXM1 + sh-KIF4 A xenografts expressed lower levels of KIF4 A as well as Ki67,a proliferation marker,as determined by immunohistochemistry and western blotting.In addition,overexpression of FOXM1 increased the protein levels of KIF4 A as well as CENPF and CCNB1,which is consistant with the in vitro experiments.Conclusions: 1.FOXM1 and KIF4 A are abnormally co-overexpressed in HCC tissues and associated with poor prognosis of HCC patients.2.KIF4 A is a direct downstream target gene of the transcription factor FOXM1 and FOXM1 regulates the expression of KIF4 A by directly binding to the promoter of KIF4 A.3.KIF4 A is required for FOXM1-mediated HCC cell proliferation.