The Critical Role Of Dysregulated Hh-FOXM1-TPX2 Signaling In Hepatocellular Carcinoma Cell Proliferation

Background:Liver cancer is one of the most frequent and difficult to treat types of malignant tumor in the world,of which hepatocellular carcinoma(HCC)accounts for more than 85%-90%.HCC has characteristics of hidden onset,difficult early diagnosis,rapid proliferation,insensitivity to conventional radiotherapy and chemotherapy,and rapid recurrence and metastasis soon after operation.Partial hepatectomy and liver transplantation offer the opportunity of cure to patients in early stage of HCC.However,the specific mechanism is not clear yet.Therefore,in-depth study of the molecular mechanisms underlying the phenotypes of HCC,such as occurrence,development,drug resistance and recurrence,and search for key molecular targets will help to achieve early screening,early diagnosis and accurate medical treatment of patients with HCC,which is of great transformational significance and is the core scientific problem to be solved urgently in the basic research field of HCC.At present,malignant proliferation of tumors is mainly attributed to a series of abnormal gene expression and regulatory networks.Abnormally activated Hedgehog(Hh)signaling pathway plays an important role in the malignant proliferation of HCC.As a key intermediate molecule,terminal effector GLI2 binds to target gene motif sequence 5'-GACCACCCA-3'when activated by upstream Hh signal,transcriptional regulates the expression of downstream cell cycle-related target genes,such as FOXM1,N-Myc,Cyclin D1,Cyclin D2,Cyclin E and so on,thus affecting mitosis and cell proliferation.However,the regulation mechanism of cell cycle is very delicate and complex.The specific molecular mechanism of Hh signaling pathway affecting the proliferation of HCC cells needs to be further explored.One of the basic characters of cancer is the disorder of cell cycle regulation mechanism,which leads to uncontrolled proliferation of cancer cells.Cell cycle related protein TPX2 plays an important role in the malignant proliferation of cancer cells.Through the analysis of gene chip expression profile data,we speculate that TPX2 is the target gene of Hh.Whether Hh signaling pathway promotes malignant proliferation of HCC cells by regulating and depending on the expression of cell cycle related protein TPX2 is the focus of this study.The purpose of this study is to elucidate the molecular mechanism of Hh signaling pathway regulating TPX2 by bioinformatics,molecular biology,cell biology and clinical cohort studies,and to demonstrate the important role of Hh-TPX2 signaling axis in regulating the malignant proliferation of HCC cells and its significance as a target.This study will help to clarify the molecular mechanism of malignant proliferation of HCC,and provide important theoretical basis for molecular diagnosis and individualized diagnosis and treatment of HCC.Materials and methods: 1.Analysis microarray or high-throughput sequencing and validate the potential target genes(1)On the one side,HCC-LM3 cells were treated with Cyclopamine(an Smo inhibitor of Hh signaling pathway),and GANT61(a GLI inhibitor).Microarrays were made to analyze gene expression profiles and identify target gene clusters that might be regulated by Hh signaling pathway.On the other side,Huh7 and HepG2 cells were treated with GANT61,and the different expressed genes were analyzed with the next generation sequence(NGS).The down-regulated genes were intersected with the cell cycle and mitosis-related gene sets in the GeneCards public database to identify target genes related to cell cycle,mitosis and regulated by Hh signaling pathway.(2)To verify the reliability of the microarray and NGS,Hep3 B,a HCC cell line with aberrant activation of Hh signaling pathway,was selected.After treatment with Cyclopamine and GANT61,the expression of TPX2,PTCH1 and FOXM1 were detected.(3)To further explore whether TPX2 is the downstream target gene of Hh signaling pathway,lentiviruses interfering with GLI2 expression were infected in Hep3 B cells.Lentiviruses overexpressing GLI2(GLI2A)were infected in Huh7 or treated with N-Shh conditional medium.The expression of TPX2,GLI2,PTCH1 and FOXM1 were detected respectively.2.To explore whether TPX2 was the downstream effector of Hh signaling pathway to accelerate the HCC cell proliferation.(1)Lentivirus system was used to construct stable HCC lentivirus cell lines in which TPX2 was overexpressed or knocked down.(2)In order to determine whether TPX2 is regulated by canonical Hh signaling pathway and promotes the malignant proliferation of HCC cells,GANT61 was added to stable HCC cell lines over-expressing TPX2,or fresh N-Shh conditional medium was added to stable HCC cell lines interfering with TPX2.EdU test,soft agar cloning formation,plate cloning formation,cell growth curve experiments and other common cell function experiments were used to explore whether Hh signaling pathway can promote malignant proliferation of HCC cells by regulating TPX2.3.Molecular Mechanisms of Hh Signaling Pathway Regulating TPX2(1)Predict the potential transcription factors upstream of TPX2 by bioinformatics tools,and predict the potential binding sites of GLI2 and FOXM1 in the promoter region of TPX2 gene.(2)Over-expression or interference of FOXM1 expression in HCC cells,and the expression of TPX2 was detected to preliminarily verify the relationship between FOXM1 and TPX2.In vivo experiment,after knocking down FOXM1,the expression of TPX2 was also detected to further confirm the regulatory relationship between them.(3)Construct TPX2 promoter region cloning of dual luciferase reporter gene,and verify whether TPX2 is the direct target gene of transcription factor FOXM1 by dual luciferase reporter gene system and chromatin immunoprecipitation.4.Verify the regulation of TPX2 by Hh-FOXM1 signal axis to promote HCC proliferation(1)TPX2 was detected by western blot in HCC cells interfering with FOXM1 as well as adding fresh N-Shh conditional medium.(2)TPX2 was detected by western blot in HCC cells with over-expression of FOXM1 while GANT61 was also added.(3)TPX2 was detected by western blot after interfering with FOXM1 expression in HCC cells overexpressing GLI2 A.(4)Hh-FOXM1 signal axis influences the malignant biological behavior of HCC by regulating the expression of TPX2.Firstly,the following stable cell lines of HCC lentiviruses were constructed to interfere with the expression of TPX2 on the basis of over-expression of FOXM1.The significance of FOXM1-TPX2 signal axis on the biological behavior of malignant proliferation of HCC cells was explored through a series of cell experiments in vitro,such as EdU,plate cloning,continuous cell counting and cell cycle detection.5.Validation of clinical cohort studies(1)Western blot and immunohistochemical staining were used to analyze the expression of FOXM1 and TPX2 in human HCC tissues and corresponding normal tissues.(2)The results of immunohistochemistry were scored by German semi-quantitative system.The correlation between the expression of FOXM1 or TPX2 and the clinicopathological characteristics of HCC patients was analyzed by chi-square test or Fisher's exact probability chi-square test.Pearson rank correlation coefficient was used to analyze the correlation between the expression of FOXM1 and TPX2 in HCC tissues.Kaplan-Meier survival curve and Log rank test were used to analyze the influence of FOXM1 and TPX2 expression levels on survival and prognosis of HCC patients.(3)Verify the relationship between the expression of FOXM1 and TPX2 and the prognosis of HCC patients in several large public databases.Results: 1.TPX2 is the downstream target gene of Hedgehog signaling pathway(1)After inhibiting Hh signaling pathway with Cyclopamine and GANT61,the expression profiles of 202 genes were analyzed by oligonucleotide microarray.The results showed that the expression of 202 genes was down-regulated(DiffScore<-50).Besides,Huh7 and HepG2 cells were also treated with GANT61,and the different expressed genes were analyzed using the next generation sequence.We found that 1711 genes were down-regulated.622 genes related to cell cycle and mitosis were crossed and many target genes were obtained,among which TPX2 belonged to microtubule-associated protein,which was related to cell mitosis and cell cycle progression.FOXM1,CDC25 B and KIF20 A were known downstream target genes of Hh signaling pathway,and could be used as positive control in the experiments;(2)After treatment with Cyclopamine,GANT61 or infection with sh-GLI2 lentivirus,the mRNA and protein level of TPX2 gene in Hep3 B cells decreased significantly,while the mRNA and protein level of TPX2 gene in Huh7 cells increased significantly after treatment with N-Shh stimulant or infection with Lv-GLI2 A lentivirus.2.TPX2 is the downstream effector of Hh signaling pathway to accelerate the HCC cell proliferation.(1)After over-expression of TPX2,cell proliferation was promoted,and after interference with TPX2,cell proliferation was significantly inhibited.(2)After interfering with TPX2,HCC cells were treated with N-Shh stimulation solution,and the proliferation of HCC cells was not significantly promoted.(3)After overexpression TPX2,GANT61 was used to inhibit Hh signaling pathway,and the proliferation of HCC cells was not inhibited.3.Hh signaling pathway regulates TPX2 expression through transcription factor FOXM1(1)The online analysis of UCSC website indicated that FOXM1 might be one of the candidate transcription factors of TPX2.Bioinformatics online analysis showed that no potential binding site of transcription factor GLI2 was found in the promoter region of TPX2.However,many potential binding sites of transcription factor FOXM1 were found.(2)It was preliminarily confirmed that there was a regulatory relationship between FOXM1 and TPX2.Overexpression of FOXM1 increased the expression of TPX2 and decreased the expression of TPX2 after interference with FOXM1.Furthermore,an regulatory relationship was confirmed between FOXM1 and TPX2 in vivo experiments.(3)The results of dual luciferase reporter system showed that FOXM1 combined with TPX2 promoter could regulate the expression of TPX2 at the transcriptional level.Through further plasmid fragmentation and binding site mutation experiments,three binding sites,FBS-9,FBS-10,FBS-11,which may play a key role,were identified.Chromatin immunoprecipitation directly confirmed that FBS-11 is the most critical binding site of FOXM1.4.Hh-FOXM1 signal axis regulates the expression of TPX2(1)To determine whether N-Shh regulates the expression of TPX2 through FOXM1,the expression of FOXM1 was disturbed in the presence of N-Shh.Western bolt was used to detect the expression of TPX2 protein,and the expression of TPX2 was decreased.(2)To further verify TPX2 downstream of the GLI2-FOXM1 signal axis,GANT61 was added to the stable cell line of HCC over-expressing FOXM1 to block the upstream Hh signal activity.It was found that the downstream over-expressing FOXM1 could rescue the upstream partially blocked Hh signal activity,and the expression of TPX2 would not be significantly inhibited by the presence of GANT61.(3)Continue to verify TPX2 downstream of GLI2-FOXM1 signal axis by blocking experiment.In Huh7 cells overexpressing GLI2 A,the expression of FOXM1 downstream was interfered and TPX2 protein was detected.It was found that the expression of TPX2 increased after overexpressing GLI2 A,but the expression of TPX2 was inhibited by interfering with FOXM1 on the basis of overexpressing GLI2 A.The upstream and downstream relationship of Hh-GLI2-FOXM1-TPX2 signal axis is further verified.(4)Compared with the control group,after interfering with TPX2 expression,the percentage of EdU positive cells in HCC cells decreased significantly,and the ability of colony formation,cell growth and globulation decreased significantly.The percentage of EdU positive cells,proliferation ability,colony forming ability,cell growth ability and globulation ability of HCC cells overexpressing FOXM1 were significantly increased.On the basis of over-expression of FOXM1,TPX2 expression downstream was blocked.Compared with over-expression of FOXM1,the percentage of EdU-positive cells decreased,and the proliferation,clone formation,cell growth and globulation ability were significantly inhibited.(5)Cell cycle experiment showed that Lv-FOXM1+sh-Control group distributed more in S phase cells than control group Lv-Control+sh-Control group,and on this basis,the effect of FOXM1 on promoting cell cycle progression was significantly inhibited after interfering with TPX2 expression.Specifically,S phase cells decreased,and G2/M phase blockade and polyploid cell formation occurred.5.The abnormal high expression of FOXM1 and TPX2 is related to the poor clinical prognosis of HCC patients(1)Western blot and immunohistochemical staining showed that the expression of FOXM1 and TPX2 in HCC tissues was significantly higher than that in the corresponding normal tissues adjacent to the cancer,which was verified by data from public database website.(2)Verify that FOXM1 is positively correlated with TPX2 expression in HCC tissues and HCC cell lines,and validate with large data from large public database websites.(3)High expression of FOXM1 or TPX2 was associated with larger tumor diameter,poorer pathological grade and higher TNM stage.(4)The total survival time and disease-free survival time of patients with liver cancer with high expression of FOXM1 and TPX2 were relatively shorter.Conclusions:1.Inhibiting Hh signaling pathway can significantly inhibit the expression of TPX2,which is the downstream target gene of Hh signaling pathway.2.Hh signaling pathway promotes the proliferation of HCC cells by regulating the expression of downstream effector molecule TPX2.3.TPX2 is the direct target gene of transcription factor FOXM1,and FOXM1 directly regulates TPX2.4.Hh-FOXM1 signal axis-mediated proliferation of HCC cells depends on the expression of TPX2.5.The expression of FOXM1 and TPX2 was abnormally high in HCC.The expression of FOXM1 and TPX2 was positively correlated with the poor prognosis of HCC.It is suggested that FOXM1 and TPX2 may be potential prognostic markers and therapeutic targets for HCC.