| Background Neuroblastoma(NBL)is the most common extracranial malignant solid tumor in children,accounting for 7-8% of malignant tumors,and 15% of cancer deaths in children.The tumor originated from the neural crest progenitor cells of the sympathetic nervous system.Tumors in high-risk NBL patients are often prone to metastasis,and about 50% of the patients have metastases at the time of diagnosis,mainly involving bone marrow,bone and regional lymph nodes.At present,the diagnostic methods are mainly based on the combination of laboratory examination,radiography and pathological diagnosis.Over the past 20 years,the overall survival rate of high-risk patients has improved,while refractory or recurrent NB patients are rarely cured.At present,the methods of radiotherapy and chemotherapy used in clinic also have great side effects on patients.There is an urgent need to find new early diagnostic markers or new safe and effective treatments.Understanding the complex molecular characteristics and genetic variation involved in the pathogenesis of neuroblastoma is necessary to develop safer and more effective treatments.Forkhead box protein M1(FOXM1),is one of the members of the FOX transcription factor family,also a proliferation-related transcription factor.It plays an important role in many biological functions,such as cell proliferation,DNA damage and repair,angiogenesis,tissue regeneration,apoptosis,tumor cell invasion and migration,and tumor chemotherapeutic drug resistance.FOXM1 has been found to be significantly overexpressed in a variety of tumors,but its molecular mechanism in NBL is rarely reported.Exploring the molecular mechanism of the occurrence and development of FOXM1 and NBL will help to find new molecular targets for the diagnosis and treatment of NBL,and lay a theoretical foundation for NBL to find new targeted therapeutic drugs.Part Ⅰ Multifaceted data to explore the effect of transcription factor FOXM1 expression in human neuroblastomaObjectiveIn this part,firstly,the differentially expressed genes in NBL were obtained by computational biology,and the expression profiles of FOXM1 in different data sets were extracted,the relationship between FOXM1 expression profiles and clinicopathological parameters of NBL patients were also analyzed.At the same time,the expression level of FOXM1 protein in clinical paraffin samples was detected to clarify the expression of FOXM1 in NBL and the relationship between FOXM1 and clinicopathological parameters.Materials and Methods1.The expression profiles and clinicopathological parameters of NBL were collected from tumor sequencing database Therapeutically Applicable Research To Generate Effective Treatments(TARGET),human normal tissue sequencing database Genotype-Tissue Expression(GTEx)and Gene Expression Omnibus(GEO)gene expression comprehensive map database.The differences of m RNA expression were analyzed by using the Voom function of R software "limma" package(for sequencing data)and "limma" package(for chip data),respectively.Then FOXM1 expression profiles were extracted.Meta-analysis was performed by Stata 14.0,and the differential expression of FOXM1 between the two groups was analyzed by Independent Sample t-test.2.The expression of FOXM1 protein in clinical paraffin samples was detected by immunohistochemical technique,and the immunohistochemical results were analyzed by Mann-Whitney U nonparametric test.Results1.FOXM1 m RNA was highly expressed in NBL tissues.A total of 6652 NBL samples and 16088 normal control samples were included in 12 chip data sets and 1 sequencing data set.After variance analysis,348 m RNA were significantly upregulated and 303 m RNA were significantly down-regulated in NBL.in addition,1477 prognostic risk factors and 1936 prognostic protective factors,as well as 2487 chemoresistance-related genes and 2899 chemosensitivity-related genes were obtained.Another 190 m RNA were significantly up-regulated and 422 m RNA were significantly down-regulated in MYCN-amplified samples.Among them,FOXM1 was significantly highly expressed in NBL(SMD = 1.76,CI = 1.15-2.38).2.FOXM1 m RNA has a good ability to distinguish NBL from normal tissue,which is significantly correlated with many clinical parameters,and the high expression of FOXM1 indicates a poor prognosis.The area under the working characteristic curve of the total subjects by FOXM1 expression profile metaanalysis was 0.94(95%CI = 0.92-0.96),suggesting that FOXM1 has a good ability to distinguish tumor tissue from normal control.At the same time,FOXM1 was significantly highly expressed in over 18-month age group,MYCN amplification group,INSS high staging group,low differentiation group and COG high risk group(all p<0.05).The event-free survival and overall survival of patients with high expression of FOXM1 were lower than those with low expression of FOXM1,suggesting that FOXM1 is a poor prognostic factor for patients with NBL.3.FOXM1 protein expression was positive in NBL paraffin specimens.The tissues of 3 cases of ganglioneuroma,9 cases of ganglioneuroblastoma and 54 cases of neuroblastoma were detected.The results showed that the expression of FOXM1 in NBL was higher than that in ganglioneuroma,and the expression trend was consistent with that of m RNA,and the difference was statistically significant(p=0.026).Conclusion1.The high expression of FOXM1 m RNA in NBL tissues was positively correlated with the clinical parameters such as age,INSS stage,MYCN amplification status,INRG risk stratification and tumor differentiation.The high expression of FOXM1 indicates a poor prognosis in patients with NBL.2.The expression of FOXM1 protein in NBL tissues was higher than that in noncancer tissues.Part Ⅱ Effects of transcription factor FOXM1 on proliferation,cycle,apoptosis,migration and invasion of neuroblastoma cells in vitroObjectiveThe purpose of this study is to verify the changes of proliferation,cycle,apoptosis,migration and invasion of NBL cells before and after FOXM1 silencing or overexpression in vitro,aiming to clarify the effect of FOXM1 on the biological behavior of NBL cells in vitro.Materials and MethodsThe expression of FOXM1 in NBL cells was detected by RT-q PCR,and the efficiency of FOXM1 silencing and overexpression was verified.The effects of FOXM1 silencing and overexpression on the proliferation of NBL cells were detected by CCK-8 assay and clone formation assay.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by wound healing and transwell assay,and cell invasion were detected by transwell experiment.Independent Sample t-test was used for comparison.Results1.FOXM1 can promote the proliferation of FOXM1 cells in vitro.1)The results of CCK-8 assay showed that when FOXM1 was silenced,the proliferation of the two NBL cells was significantly inhibited,compared with the negative control group.On the contrary,the proliferation ability of NBL cells was significantly higher than that of the negative control after overexpression of FOXM1.2)The results of clone formation assay showed that the clone formation rates of the two negative control groups were 19.3%(for si-FOXM1 group)and 16.3%(for OE-FOXM1 group),respectively,while the clone formation rates of si-FOXM1-1 group,si-FOXM1-2 group and OE-FOXM1 group were 14.1%,15.5% and 35.9%,respectively.It is suggested that FOXM1 can affect the proliferation of NBL cells in vitro.2.FOXM1 can affect the migration and invasion of NBL cells in vitro.1)The results of wound healing assay showed that after silencing FOXM1 the healing ability of the two cell lines at 24 h and 48 h was significantly lower than that in vector group(p<0.05).When FOXM1 was overexpressed,the healing ability of NBL cells was significantly higher than that in vector group(p<0.05).2)The results of Transwell chamber experiment showed that compared with Vector group,the number of NBL cells passing through compartment polycarbonate membrane significantly decreased after silencing FOXM1 expression(p<0.001),while FOXM1 overexpression significantly improved the ability of NBL cells to pass through compartment polycarbonate membrane,and the number of cells passing through compartment in FOXM1 overexpression group was significantly higher than that in Vector group(p<0.001).3)when FOXM1 was silenced,the number of SK-N-BE(2)cells in si-FOXM1-1 group and si-FOXM1-2 group passing through the membrane of matrigel matrix glue was significantly lower than that in Vector group(p<0.0l).When FOXM1 was overexpressed,the number of cells penetrating the membrane was significantly increased compared with that of Vector group,and the invasive ability of SK-N-BE(2)cells was significantly improved(p<0.001),suggesting that FOXM1 can also significantly promote the invasive ability of NBL cells.3.Silencing the expression of FOXM1 in vitro could block NBL cells in G2/M phase.The effect of FOXM1 on NBL cell cycle was detected by flow cytometry.The results showed that compared with Vector group,the percentage of G0/G1 phase cells in si-FOXM1-1 group and si-FOXM1-2 group was significantly decreased,while the proportion of G2/M phase cells was significantly increased.On the contrary,after the overexpression of FOXM1,the proportion of cells in G0/G1 phase increased,while the proportion of cells in G2/M phase decreased,the proportion of cells in S phase had no significant change(p<0.05).It is suggested that after FOXM1 silencing,NBL cells are blocked in G2/M phase,thus inhibiting the proliferation of NBL cells.4.Silencing the expression of FOXM1 in vitro can induce apoptosis of NBL cells.The effect of FOXM1 on apoptosis of NBL cells was detected by flow cytometry.The results showed that the proportion of early apoptotic cells and late apoptotic cells in si-FOXM1-1 group and si-FOXM1-2 group were higher than those in Vector group,but the difference was not significant.However,the proportion of early and late apoptosis in FOXM1 overexpression group was significantly lower than that in Vector group(p<0.05).It is suggested that silencing FOXM1 in vitro can induce apoptosis of NBL cells.Conclusion1.FOXM1 can promote the proliferation,migration and invasion of NBL cells,while inhibit the apoptosis of NBL cells in vitro.Part Ⅲ Screening potential targets for the role of FOXM1 in neuroblastoma based on high-throughput sequencingObjectiveIn this part,we use chromatin co-immunoprecipitation technique(Ch IP-seq)to specifically enrich the DNA sequence bound by FOXM1 in NBL,and transcriptome RNA-seq sequencing of NBL cells before and after FOXM1 interference to explore the potential target genes of FOXM1 in NBL,as well as the metabolic pathways of gene ontology(GO)and Kyoto Encyclopedia of Gene and Genome(KEGG)involved in them,aiming to analyze their possible biological functions.Materials and MethodsTwo pairs of FOXM1-bound and unbound NBL cell samples were collected and sequenced by Ch IP-seq,3 pairs NBL cell samples with before and after FOXM1 interference were sequenced by RNA-seq,the peak enriched by Ch IP-seq was annotated and visualized by R software "Ch IPseeker" package,and the differentially expressed m RNA was obtained by R software "edge R" package.The R software "cluster Profiler" package was used to annotate the GO function and KEGG pathway of differential expression genes.The target genes of FOXM1 were predicted by transcription factor prediction website h TFtarget,and the potential target genes of FOXM1 in NBL were screened.Results1.The distribution of Ch IP-seq peak in SK-N-BE(2)cells and SK-N-SH cells on chromosomes and the distribution of the peak relative to the gene position.Compared with SK-N-SH cells,most of the peaks of SK-N-BE(2)cells were distributed in the remote intergene region(36.34%)and intron region(32.74%),and the peak distribution in the range of 3kb around the promoter is 12.03%.The peak of FOXM1 in SK-N-SH cells was mainly in the range of 1kb around the promoter(29.1%),and a small part of FOXM1 was distributed in the range of 1-3kb(6.12%),followed by other intron regions except the first intron(25.05%)and distant genes(23.67%).2.Enrichment analysis of GO and KEGG with genes near the peaks.57 significant biological process(BP)items were enriched by GO analysis of potential target genes of FOXM1.The first five most significant items are DNA replication-dependent nucleosome assembly,DNA replication-dependent nucleosome organization,negative regulation of gene expression,epigenetic,r DNA heterochromatin assembly and chromatin organization involved in negative regulation of transcription.KEGG pathway is enriched into seven significant metabolic pathways,among which Viral carcinogenesis and cell cycle are closely related to tumorigenesis.3.The KEGG metabolic pathway enriched with differential expression m RNA.A total of 496 up-regulated genes and 509 down-regulated genes were obtained(p<0.05).KEGG metabolic pathway analysis showed that differentially expressed genes were significantly enriched in 19 KEGG metabolic pathways.Among them,the first five metabolic pathways with the most significant enrichment are PI3K-Akt signaling pathway,TGF-beta signaling pathway,Calcium signaling pathway,Proteoglycans in cancer and ECM-receptor interaction,are all significantly associated with tumorigenesis.4.Screening of potential differentially expressed targets of FOXM1 in NBL.HTFtarget website estimated that 1733 potential target genes of FOXM1 were obtained.Four potential FOXM1 target genes,NAV2,NEURL1 B,NEK2,and TGFBI,were obtained by intersecting with the target genes obtained by Ch IP-seq analysis and m RNA which were significantly differentially expressed before and after FOXM1 silencing.The transcriptional direction of NEK2 was the same as that of FOXM1,and was significantly up-regulated in NBL.Conclusion1.FOXM1 may regulate the progression of NBL through PI3K-Akt signal pathway,TGF-beta signal pathway,calcium signal pathway,proteoglycan and ECM-receptor interaction in cancer.2.NAV2,NEURL1 B,NEK2 and TGFBI genes may be potential targets of FOXM1 in NBL.Part Ⅳ FOXM1 promotes the progression of neuroblastoma by targeting NEK2ObjectiveIn this part,we combined with high-throughput chip sequencing data,immunohistochemical results and cellular level RT-q PCR and western blot to verify the expression level of NEK2 in NBL,and to verify the targeted binding relationship between NEK2 and FOXM1.Materials and MethodsIndependent Sample t-test was used to analyze the expression level of NEK2 in high-throughput chip sequencing data set and further verified by meta-analysis.Pearson correlation test was used to analyze the correlation between NEK2 and FOXM1.Immunohistochemical technique was used to detect the protein expression level of NEK2 in clinical samples of NBL.The expression level of NEK2 before and after FOXM1 silencing was verified by RT-q PCR and Western blot techniques.Finally,the targeted binding relationship between FOXM1 and NEK2 was verified by double luciferase reporting assay.Results1.High-throughput sequencing and chip data confirmed that NEK2 m RNA was significantly overexpressed in NBL tissues and significantly correlated with multiple clinical parameters.The results of meta-analysis showed that SMD=1.58(95%CI:0.97-2.18),suggesting that the expression level of NEK2 in NBL was significantly higher than that in normal control.The area under the SROC curve was 0.94(95%CI = 0.92-0.94),suggesting that NEK2 had a good ability to distinguish NBL from non-cancer tissues,and the expression of NEK2 was significantly higher in over 18-month age group,MYCN amplification group,INSS high staging group,low differentiation group and COG high risk group(all p<0.05).The event free survival and overall survival of patients with high expression of NEK2 were lower than those of patients with low expression of NEK2,suggesting that NEK2 is a poor prognostic factor for NBL.2.The results of immunohistochemistry showed that NEK2 protein expression was positive in NBL tissue.The protein expression level of NEK2 in 5 cases of ganglioneuroma,9 cases of ganglioneuroblastoma and 54 cases of neuroblastoma showed that the expression of NEK2 in NBL was significantly higher than that in ganglioneuroma(p=0.014).In addition,the expression level of NEK2 protein in III-IV stage of INSS stage was significantly higher than that in stage I-II(Z=-1.969,p=0.049).3.Silencing the expression of FOXM1 could significantly down-regulate the expression of NEK2.RT-q PCR results showed that the m RNA expression of NEK2 in FOXM1 silence group was significantly lower than that in vector group(p<0.05).Western blot results revealed that,the protein expression of NEK2 in FOXM1 silence group was lower than that in vector group.4.The expression level of NEK2 was positively correlated with that of FOXM1.The results of correlation analysis between the expression of NEK2 and FOXM1 showed that there was a significant positive correlation between NEK2 and the expression of FOXM1.5.There is a targeted binding relationship between FOXM1 and NEK2 promoter.HTFtarget website predicted the binding sites of FOXM1 and NEK2 promoters online showed that there were 7 potential targeted binding sites between FOXM1 and NEK2 promoters(prediction score > 10,and Q value < 0.05).and the double luciferase report assay further confirmed that FOXM1 could bind to the NEK2 promoter.Conclusion1.The high expression of NEK2 in NBL was positively correlated with patient’s age,MYCN amplification status,INSS stage and INRG risk stratification,and negatively correlated with tumor differentiation.And the high expression of NEK2 indicates a poor prognosis of the NBL patients.2.There is a targeted binding relationship between FOXM1 and NEK2 promoter.Part Ⅴ The regulatory axis of FOXM1 targeting NEK2 to promote the progression of neuroblastoma can be inhibited by nitidine chloride in vitroObjectiveIn this part,we conducted molecular docking of NC with FOXM1 and its target gene NEK2,and found that NC had a strong affinity with NEK2.Hence,we used human neuroblastoma SK-N-BE(2)cells as a model to explore the effects of nitidine chloride(NC)on cytotoxicity,cycle,apoptosis and migration of NBL cells in vitro.The purpose of this study is to lay a preliminary theoretical foundation for further exploring the potential molecular mechanism of NC antiNBL,and to provide a theoretical basis for clinical patients with NBL who are ineffective by conventional medication to find new alternative treatment strategies.Materials and MethodsThe targeted binding relationship between FOXM1 and NEK2 and NC was predicted by molecular docking technology.The expression levels of FOXM1 and NEK2 in cells were detected by RT-q PCR,the effects of NC on the proliferation of NBL cells were detected by CCK-8 assay,cell cycle and apoptosis were detected by flow cytometry,and NBL cell migration were detected by wound healing assay.Results1.Molecular docking results suggest that there may be a targeted binding relationship between NEK2 and NC.Molecular docking results showed that the docking score between NEK2 and NC was 5.592,suggesting that NEK2 had a strong affinity with NC.NC may act indirectly by directly targeting the binding of NEK2 to FOXM1.2.NC can inhibit the proliferation of NBL cells in vitro.The results showed that NC had obvious cytotoxic effect on NBL cells in a certain concentration range,and could significantly inhibit the cell viability,and the inhibition of NC on the activity of SK-N-BE(2)and SK-N-SH cells was time-and concentrationdependent.The IC50 value of SK-N-BE(2)cells treated with NC for 24 h was 4.177μmol/L and the IC50 value of SK-N-SH cells treated with NC for 24 h was 9.105μmol/L.3.NC blocked SK-N-BE(2)cells in S phase in vitro.Compared with DMSO group,the number of cells in G1 phase decreased while the number of cells in S phase increased significantly in NC group after treated with 4μmol/L NC for 24 h.The results suggested that NC may inhibit the proliferation of SK-N-BE(2)cells by blocking it at S phase.4.NC induced apoptosis of SK-N-BE(2)cells in vitro.The results showed that the proportion of early apoptotic cells in the experimental group with 4μmol/L NC(2.395±0.120%)was lower than that in the DMSO group(3.290 ±0.834%),but the difference was not statistically significant(p=0.272).The proportion of late apoptotic cells(13.65 ±2.616%)was significantly higher than that of DMSO group(1.77±0.014%)(P = 0.023).The proportion of total apoptotic cells(16.045 ±2.496%)was significantly higher than that of DMSO group(5.06 ±0.820%)(p=0.027).5.NC inhibited the migration of SK-N-BE(2)cells in vitro.The effect of NC on the migration of SK-N-BE(2)cells was detected by wound healing assay.The results showed that the migration ability of SK-N-BE(2)cells treated with 4μmol/L and 8μmol/L NC for 24 h and 36 h was significantly lower than that of DMSO group,which suggested that NC could inhibit the migration of SK-N-BE(2)cells in vitro.6.NC can inhibit the expression of FOXM1 in vitro.RNA was extracted from SK-N-BE(2)cells treated with DMSO,4μmol/L NC and 8μmol/L NC for 48 h,and the m RNA expression levels of FOXM1 and NEK2 were detected by RTq PCR.The results showed that compared with DMSO group,the expression level of FOXM1 decreased significantly after 48 h of NC administration,and the down- regulation level of FOXM1 was positively correlated with the degree of NCadministration.Conclusion1.There may be a direct targeting binding relationship between NEK2 and NC.2.NC can inhibit the proliferation and migration of NBL cells in vitro,block the NBL cell cycle in S phase,and induce NBL cells apoptosis.3.NC can inhibit the expression of FOXM1 in vitro. |
PDF Full Text Request