The Study Of The Role And Pathogenic Mechanism Of Transcript Factor FOXN3 In Acute Myeloid Leukaemia

Objective : Acute myeloid leukaemia(AML),the most common type of adult leukaemia,is characterized by out-of-control proliferation,the inhibition of differentiation,the apoptotic blockage of leukocytes,and reduction in normal haematopoietic cells.Although AML could be treated with a better therapeutic effect,60%-80% of AML patients could not be cured due to disease resistance and recurrence.Heredity,radiation,chemical and other occupational exposures,and drugs have been implicated in the development of AML,the exact etiology of most AML is not clear.Therefore,it is always a difficult medical question to study the pathogenesis and potential therapeutic strategy for AML.With the development of cytogenetic and molecular biology,abnormal structure or number of chromosomes,gene mutation and abnormal expression of oncogenes or tumour suppressor genes are considered to be related with the leukaemogenesis of AML.Recent studies that focused on abnormal transcription demonstrate the key roles of transcription regulators in leukaemogenesis and suggest a potential therapeutic strategy for AML.Therefore,the identification of novel abnormal transcription factors and their functions in AML will provide new clues on the pathogenesis and treatment of AML.The transcription factor forkhead box N3(FOXN3),also known as checkpoint suppressor 1(CHES1),as a member of the forkhead box N superfamily,participates in several biological processes,including the cell cycle,cell differentiation,epithelialmesenchymal transition,gene transcription and glucose metabolism and so on.It has shown in previous studies that the downregulated expression of FOXN3 is observed in several kinds of various malignancies,such as hepatocellular carcinoma,lung cancer,colon cancer,prostate cancer,head and neck cancer,adult glioblastoma multiforme,lymphoma,and so on,and its downregulation could promote the progression of cancer,these indicate that FOXN3 might be an important tumour suppressor gene.However,the mechanism of FOXN3 in cancers is still poorly understood.On one kind,FOXN3 has been reported to inhibit the expression of some tumour oncogenes.On the other kind,FOXN3 has also been reported to bind with several proteins.However,the biological function of FOXN3 on AML remains largely unknown.The transcription factor E2 F transcription factor 5(E2F5)as a member of the E2 F family,which participate in several biological processes including the cell proliferation,cell cycle,DNA damage repairment and development.E2F5 plays an oncogene role in various solid tumours such as hepatocellular carcinoma,prostate cancer,ovarian cancer and esophageal squamous cell carcinoma,and is related to the sensitivity of chemotherapy.To the best of our knowledge,the expression of E2F5 in AML was upregulated than control in only one previous report using c DNA microarray.However,its biological function on AML remains unknown.Although lower FOXN3 expression in adult AML was found in our previous study,its clinical and prognostic significance in AML remained largely unknown.Our study investigated the expression profile of FOXN3 in the bone marrow(BM)of adult AML patients and analysed its clinical significance in larger cohort of patients.We investigated the biological function and possible pathogenesis mechanism of FOXN3 in AML,which could provide evidence that FOXN3 might be a potential biomarker and therapeutic target in AML.Methods: 1.117 newly diagnosed AML patients and 25 healthy donors were collected.A total of 34 BM samples from patients with AML at the time points of complete remission(CR)were also collected.FOXN3 m RNA expression was detected by realtime quantitative polymerase chain reaction(RT-q PCR)as the following steps: extracting mononuclear cells from BM,isolating total RNA,reverse transcription to c DNA and RT-q PCR.FOXN3 expression was compared between haematopoietic stem cells(HSCs)and AML from the Bloodpool dataset using the online Blood Spot database(www.bloodspot.eu).The expression of FOXN3 protein was confirmed downregulated by streptavidin-perosidase(SP)immunocytochemical staining.The clinical features of 117 newly diagnosed AML patients such as age,sex,white blood cell,hemoglobin,platelet,conventional chromosome banding,gene mutation,therapy and survival data were collected and compared between lower FOXN3 expression group and higher FOXN3 expression group.2.AML cell lines THP1,Kasumi1,U937,HL60 and 293 T cell line were cultured.The FOXN3 expression of the above cell lines were detected by RT-q PCR and Western blot.Kasumi1 and THP1 cells were transfected with FOXN3 overexpression or sh RNA lentivirus to upregulate or knockdown the expression of FOXN3.The FOXN3 expression were detected by RT-q PCR and Western blot after FOXN3 overexpression or sh RNA lentivirus transfection.The proliferation capacity was detected by CCK-8 assay method,cell cycle and cell apoptosis were detected by flow cytometry after FOXN3 overexpression or sh RNA lentivirus transfection.3.We performed chromatin immunoprecipitation sequencing(Ch IP-seq)upon human THP1 cells using FOXN3-specific antibody to screen the potential target genes regulated by FOXN3.To understand the biological processes and pathways regulated by FOXN3,we performed gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.And the expression of the potential target genes was detected by RT-q PCR and Western blot upon FOXN3 overexpression.The online tool Jaspar(http://jaspar.binf.ku.dk/)was used to predict the potential binding sites.Chromatin immunoprecipitation-PCR(Ch IP-PCR)and dual luciferase reporter gene assay were performed to confirm that E2F5 was the target gene regulated by FOXN3 directly.AML cell lines THP1,Kasumi1,K562 and 293 T cell line were cultured.The E2F5 expression of the above cell lines and BM samples from newly diagnosed AML and healthy donors were detected by RT-q PCR.E2F5 expression was compared between HSCs and AML from the Bloodpool dataset using the online Blood Spot database(www.bloodspot.eu).Kasumi1 and THP1 cells were transfected with E2F5 sh RNA lentivirus to knockdown the expression of E2F5.The E2F5 expression was detected by RT-q PCR and Western blot after E2F5 sh RNA lentivirus transfection.The proliferation capacity was detected by CCK-8 assay method,cell cycle and cell apoptosis were detected by flow cytometry after E2F5 sh RNA lentivirus transfection.The protein levels of PCNA,Bcl-2,cyclin D1,CDK6,CDK2,cyclin E1 were detected by Western blot after E2F5 sh RNA lentivirus transfection.Kasumi1 and THP1 cells were co-transfected with FOXN3 overexpression and E2F5 overexpression lentivirus or control lentivirus,the expression of FOXN3 and E2F5 expression were detected by RT-q PCR and Western blot.The proliferation capacity was detected by CCK-8 assay method,cell cycle and cell apoptosis were detected by flow cytometry.We applied RNA-sequencing(RNA-seq)to screen the changes of m RNA gene expression profile upon FOXN3 overexpression and further performed GO and KEGG pathway enrichment analysis.The protein levels of PCNA,Bcl-2,cyclin D1,CDK6,CDK2,cyclin E1,EZH2,ERK,p-ERK were detected by Western blot.4.Statistical analysis: Data were presented as the mean ± standard deviation.Statistical analysis was performed by Graph Pad Prism 7.0a software and SPSS 15.1 software.Differences between groups were compared using the Mann-Whitney test or one-way analysis of variance(ANOVA)among multiple groups.Pearson chi-square analysis/Fisher's exact test was conducted to compare the differences of categorical variables.Survival analysis was used to analyse the impact of FOXN3 on relapse free survival(RFS)and overall survival(OS),and the differences were compared by a logrank test.Univariate and multivariate analyses were performed using the Cox promotional hazards regression model.Results were considered statistically significant when P<0.05.Results: 1.The clinical and prognostic significance of FOXN3 downregulation in AML.1.1 FOXN3 expression was abnormally down-regulated in AML(m RNA and protein level),and it changed significant higher in the CR phase.FOXN3 expression was significantly lower in AML than in HSCs.1.2 The lower expression of FOXN3 was associated with older age and higher white blood cell counts.Patients with lower FOXN3 expression had a lower CR rate and shorter OS.A significant difference in RFS between the two groups with different levels of FOXN3 expression was observed when older patients were excluded.Univariate and multivariate analyses showed that the expression of FOXN3 was an independent prognostic factor.2.Effect of FOXN3 on the biological function of AML cells.2.1 FOXN3 expression was significantly lower in AML cell lines than control.2.2 FOXN3 expression changed significantly higher after the FOXN3 overexpression lentivirus transfection into Kasumi1 and THP1 cells.FOXN3 expression changed significantly lower after the FOXN3 sh RNA lentivirus transfection into Kasumi1 and THP1 cells.2.3 FOXN3 overexpression lentivirus was transfected into Kasumi1 and THP1 cell line,CCK-8 assay showed that the cell proliferation was inhibited,flow cytometry showed that cell cycle was blocked in G0-G1 phase and cell apoptosis increased.FOXN3 sh RNA lentivirus was transfected into Kasumi1 and THP1 cell line,CCK-8 assay showed that the cell proliferation increased,flow cytometry showed that cells in G0-G1 phase reduced and cell apoptosis was inhibited.3.FOXN3 functions as a tumour suppressor in AML via inhibition of the MAPK signalling pathway by transcriptionally regulating E2F5.3.1 The target genes of FOXN3 were preliminary screened by Ch IP-Seq.We obtained 205115 binding sites for FOXN3,among which 2.49% located in promoter,1.47% located in exon,33.09% located in intron,11.09% located in promoter upstream regions.By associating each binding site with the nearest gene within 5 kb upstream and 5 kb downstream of coding region,we identified a total of 5719 FOXN3 potential target genes.These genes were enriched in the processes including cell cycle arrest,regulation of apoptotic signaling pathway,development and stress using GO analysis.And these genes were enriched in the pathways including cell cycle,adhesion junction and galactose metabolism using KEGG pathway analysis.3.2 The target genes were further screened using RT-q PCR and Western blot.The expression of CDC42,E2F5 and HDAC1 which were screened through Ch IP-seq was confirmed significantly lower in THP1 cells upon FOXN3-overexpression than negative control cells by RT-q PCR.The protein level of CDC42,E2F5 was confirmed significantly lower in THP1 cells upon FOXN3-overexpression than negative control cells by Western blot.3.3 E2F5 was identified as the transcriptional target gene of FOXN3.There were potential binding sites between FOXN3 and E2F5 using online tool Jaspar.Ch IP-PCR and dual luciferase reporter gene assay were performed to confirm the direct target relationship between FOXN3 and E2F5.FOXN3 could bind to the E2F5 promoter region directly and inhibit its transcriptional activity.3.4 E2F5 was proven to be a novel oncogene in AML.E2F5 expression was significantly higher in AML than in HSCs,E2F5 expression was abnormally up-regulated in AML,E2F5 expression was significantly higher in AML cell lines than control.E2F5 sh RNA lentivirus was transfected into Kasumi1 and THP1 cell line.The significant downregulated expression of E2F5 was detected by RT-q PCR and Western blot upon E2F5 knockdown.Upon E2F5 knockdown,flow cytometry showed that cell cycle was blocked in G0-G1 phase and cell apoptosis increased,CCK-8 assay showed that the cell proliferation was inhibited,which is consistent with the biological function of FOXN3 overexpression on leukemic cells.Western blot results showed that the protein levels of PCNA,Bcl-2,cyclin D1,CDK6,CDK2,cyclin E1 were downregulated on E2F5 knockdown.3.5 FOXN3 functioned as a tumour suppressor through transcriptionally regulating E2F5.Overexpression of E2F5 significantly rescued the effect including proliferation,cell cycle,and cell apoptosis of FOXN3 overexpression upon Kasumi1 and THP1 cells.The results of Western blot assay showed overexpression of E2F5 significantly rescued the effect of FOXN3 overexpression on the expression of PCNA,Bcl-2,cyclin D1,CDK6,CDK2,cyclin E1.3.6 FOXN3-E2F5-EZH2-MAPK axis in AML.Upon FOXN3 overexpression,we identified a total of 181 significantly differential genes(94 upregulated and 87 downregulated).These genes were enriched in the processes including regulation of transcription,cell cycle,apoptotic process using GO analysis.And these genes were enriched in the pathways including MAPK signaling pathway,Erb B signaling pathway,NF-kappa B signaling pathway.The results of Western blot assay showed overexpression of E2F5 significantly rescued the effect of FOXN3 overexpression on the expression of EZH2 and p-ERK.Conclusions: 1.Expression of FOXN3 was significantly down-regulated in AML patients and cell lines than normal control.The lower expression of FOXN3 was associated with older age,higher white blood cell counts,lower CR rate and shorter OS in AML patients.The expression of FOXN3 was an independent prognostic factor.2.FOXN3 overexpression could induce cell cycle arrest,increase cell apoptosis of AML cells,and inhibit the proliferation.FOXN3 knockdown could induce cell cycle and increase the proliferation.3.E2F5 was identified as the transcriptional target gene of FOXN3 in AML.E2F5 was proven to be a novel oncogene in AML,E2F5 knockdown could induce cell cycle arrest,increase cell apoptosis of AML cells,and inhibit the proliferation.FOXN3 functioned as a tumour suppressor in AML via FOXN3-E2F5-EZH2-MAPK axis.