Crucial Roles Of Intelectin 1 In Suppressing The Growth,Invasion,and Metastasis Of Neuroblastoma Cells And Its Underlying Mechanisms

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ITLN1 facilitates the NDRG2 expression at transcriptional levels in NB cellsObjective:Searching for target gene of ITLN1 in neuroblastoma and validate the effect of ITLN1 to the target gene.Methods:By data mining of the public available clinical tumor expressiondatasets R2:microarray analysis and visualization platform(http://hgserverl.amc.nl/cgi-bin/r2/main.cgi)including colon cancer,lung cancer,renal cancer,prostatecancer,and NB.Then confirm the candidate target genes by realtime quantative PCR,Western-blot and promoter luciferase assay to reveal the potential target of ITLN1 in neuroblastoma.Results:Multi datasets analysis reveal that the ITLN1 and NDRG2 transcript levels in NB tissues were positively correlated(p<0.05).Transfected ITLN1 plasmid markedly induced the expression of NDRG2 in NB cells(6.26 folds,p<0.01).The expression of vascular endothelial growthfactor(VEGF)and matrix metallopeptidase 9(MMP-9)was also decreased by ITLN1 transfection(0.17 fold/0.15 fold,p<0.01)or recombinant ITLN1 protein(0.23 fold/0.28 fold,p<0.01).Transfection of sh-ITLN1 into SH-SY5Y and SK-N-SH cells resulted in obviously reduced expression and secretion of ITLN1,decreased NDRG2 levels(0.24 fold,p<0.01),and increased expression of VEGF and MMP-9.Stable over-expression or knockdown of ITLN1 resulted in increased(1.88 folds,p<0.05)and decreased NDRG2 promoter activity(0.47 fold,p<0.05)in NB cellsConclusions:ITLN1 considerably facilitates the transcription and expression of NDRG2 in NB cells.Part ? Involvement of KLF4 in ITLN1-mediated up-regulation of NDRG2Objective:To investigate the mechanisms underlying ITLN1-mediated up-regulation of NDRG2,especial ly the specific transcription factor that regulates NDRG2 in neuroblastoma.Methods:By analyzing the transcription factor binding sites within NDRG2 promoter,combine with genes correlated with NDRG2 in 88 NB cases derived from R2 microarray analysis andvisualization platform.Then comfirm the hypothesis with experiments including realtime quantative PCR,Western-blot and promoter luciferase assay,chromatin immunoprecipitation(ChIP).Results:By analyzing the transcription factor binding sites within NDRG2 promoter,and noted one potential binding site of transcriptionfactor KLF4,locating at bases 7-18 upstream the TSS,data derived from R2 microarray analysis andvisualization platform also noted KLF4 correlation with NDRG2 in NB.Dual-luciferase assay indicated that ectopic expression or knockdown of KLF4 increased(6.89 folds,p<0.05)and decreased(0.27 fold,p<0.05)the promoter activity of NDRG2 in NB cells,and the ITLN1-facilitated NDRG2 promoter activity was abolished by mutation of KLF4 binding site.Chromatin immunoprecipitation and quantitative PCR indicated that over-expression or knockdown of ITLN1 increased(4.32 folds,p<0.05)or decreased(0.25 fold,p<0.05)the binding of KLF4 on-133/+55 region of NDRG2 promoter in SH-SY5Y and SK-N-SH cells,which was rescued by knockdown or ectopic expression of KLF4.ITLN1 up-regulated the protein levels,but not the transcript levels,of KLF4 in NB cellsConclusions:KLF4 facilitatesthe transcription of NDRG2,and plays a crucial rolein ITLN1-induced up-regulation of NDRG2 in NB cells.Part ? ITLN1 facilitates the expression of KLF4 via inactivation of PI3K/AKT signalingObjective:Further explore the mechanisms for ITLN1-induced KLF4 expressionMethods:By article review combine with Western-blot of candidate signaling pathways to confirmed the pathway by which ITLN1 regulates KLF4 expression.Results:Administration of ITLN1 transfection reduced the phosphorylation of AKT(T308 and S473)(0.12 fold&0.24 fold respectively,p<0.01),and up-regulated the expression of KLF4(5.24 folds,P<0.01),stable knockdown of ITLN1 induced the phosphorylation of AKT(T308 and S473)(3.35 folds&4.77 folds respectively,p<0.01),and downregulated the KLF4 expression(0.16 fold,P<0.01).Administration of PI3K activity inhibitor LY294002 will abolish the effects of ITLNI konckdown(p<0.05).Conclusions:ITLN1 facilitates the KLF4 expressionthrough attenuating PI3K/AKT signaling in NB cells.Part IV Ectopic expression of ITLN1 suppresses the growth,migration and invasion of NB cells through up-regulating NDRG2Objective:Investigate the effects of ITLN1 overexpression and target gene restoration on cultured NB cells,in contrast,investigate the effects of ITLNI knockdown and NDRG2 restoration on cultured NB cells.Methods:MTT colorimetric assay,colony formation assay,scratch assay,transwell analysis of ITLN1 stable overexpression and knockdown celllines together with NDRG2 restoration to explore the tumorigenicity of NB celllines.In vivo experiments include subcutaneous xenograft tumors in athymic nude mice and metastasis nude mice with tumor injected into tail vein.Then harveste and staine with HE.Results:NB cells stably transfected with ITLN1 presented an impaired invasion capacity in transwell assay(p<0.05),attenuated the growth of NB cells in MTT and colony formation assay(p<0.05),attenuated the migration capabilities of NB cells in migration assay(p<0.05).Restoration of NDRG2 expressionrescued the NB cells from their changes in thesephenotypes induced by stable over-expression of ITLN1(p<0.05).In contrast,in MTT colorimetric and colony formation assays,knockdown of ITLN1 facilitated their viability and growth(p<0.05),increased the migration capabilities in scratch assay(p<0.05)and in the mean time,restoration of NDRG2 expression rescued the NB cells from their changes in these phenotypes induced by stable knockdown of ITLN1.In vivo experiments have shown that stable transfection of ITLN1 into SH-SY5Y cells resulted in decreased growth and tumor weight of subcutaneous xenograft tumors in athymic nude mice(p<0.05).And in metastasis analysis,stable transfection of ITLN1 established fewer lung metastatic colonies than mock group(p<0.05).Stable knockdown of ITLN1 in SH-SY5Y cells resulted in increased growth and tumor weight of subcutaneous xenograft tumors in athymicnude mice and more lung metastatic colonies in metastatic analysis(p<0.05).Conclusions:ITLN1 suppresses the growth,migration,and invasion of NB cells in vitro and in vivo.Part ? ITLN1 is under-expressed and inversely correlated with NDRG2 in NB tissues and cell linesObjective:To investigate the ITLN1 and NDRG2 expression in NB clinical specimens and celllines,analyse the correlation between ITLN1 and NDRG2 transcription and expression.Methods:Paraffin-embedded sections from 42 well-established primary cases were collected,levels of ITLNland NDRG2 were evaluated by immunohistochemical staining along with differentiation,MKI index,INSS stage.Western-blot and real-time quantitative RT-PCR tested the transcription level and protein expression level of NB specimens and celllines.R2 microarray analysis and visualization platform revealed the survival probability of patients with different expression levels of ITLN1 and NDRG2.Result:ITLN1 expression was detected in 14/42(33.3%)cases and the staining was weak in 6,moderate in 5,and intense in 3,and its immunoreactivity was significantly higher in NB cases with age less than 1 year(p =0.03),good differentiation(P<0.001),lower mitosis karyorrhexis index(MK1)(p=0.002),and early INSS stages(p =0.003).Western-blot and real-time quantitative RT-PCR indicated lower expression levels of ITLN1 and NDRG2 in NB specimens and celllines than in normal dorsal ganglia.Kaplan-Meier survival plots of welldefined NB cases derived from R2 microarray analysis and visualization platform revealed that patients with high ITLN1(p = 0.025)or NDRG2(p=0.0015)expressionhad greater survival probabilityConclusion:ITLN1 is under-expressed and correlated with the NDRG2 expression in NB tissues and cell lines.Higher expression levels of ITLN1 and NDRG2 may have protective roles in NB patients.