SNW1 Regulates Notch Signaling In Neuroblastoma Through Interacting With RBPJ

Part one: Transcription factor RBPJ regulates Notch signaling in neuroblastomaObjective: To explore the crucial transcriptional factors(TF)involved in the regulation of Notch signaling process as well as its mechanisms in neuroblastoma(NB).Methods: To comprehensively investigate the key transcription factor in the Notch signaling pathway,we first analyzed the public microarray datasets derived from the Gene Expression Omnibus.Based on overlapping analysis of these TF associated with clinical progression,international neuroblastoma staging system(INSS)stage,the survival significantly TF was chosen.Western blot(WB)and immunohistochemical(IHC)staining of NB samples were performed to detect its effects on the protein levels of TF.The protein levels of TF in Ganglia tumor,well differentiated,poorly differentiated and undifferentiated NB tissues were examined to determine their potential clinical significance.Real-time quantitative PCR and Ch IP assays were performed to observe the roles of TF in transcriptional regulation of Notch signaling pathway related genes.Results: Through comprehensive analysis of GEO database(GSE62564)derived from the Gene Expression Omnibus,based on the overlapping analysis of these TF associated with clinical progression,INSS stage(P < 0.05,false discovery rate(FDR)< 0.05),we found that MAML3 and RBPJ might be a key transcription factor involved in Notch signaling pathway.According to the significance of survival,we chose the RBPJ gene for further examination.Moreover,WB and IHC staining of NB samples revealed higher RBPJ expression in non-differentiated tissue than in differentiated tissue.Depletion of RBPJ using two independent short hairpin RNA(sh RNA),sh-RBPJ#1 and RBPJ#2,in NB cell lines resulted in the upregulation of Notch target genes HES1,HES4,HEY1,and HEY2,but not of PGAM5 in NB cells.Ch IP and real-time quantitative PCR(q PCR)assays indicated the endogenous enrichment of RBPJ on the promoter regions of HES1,HES4,HEY1,and HEY2,which was decreased and by extopic knockdown of RBPJ.Conclusions: These results indicated that as a crucial transcription factor,RBPJ was able to regulate and participate in Notch signaling pathway in NB and as an independent marker of NB progression and outcome.Part two: SNW1 interact with the transcription factor of RBPJObjective: To investigate the interacting partner of RBPJ and explore function of interacting partners in NB.Methods: To determine the interacting partner of RBPJ,we analyzed the publicly available datasets Bi OGRID,IID,and STRING.Through immunoprecipitation(IP)and Coomassie blue staining,we identified the potential partners of RBPJ.In addition,we performed RBPJ/partner antibody pull-down assays and bimolecular fluorescence complementation(Bi FC)assay,suggesting that the partner interact with RBPJ.From the GEO database(GSE62564),the interacting partner of RBPJ survival curves and expression levels was analyzed in public database.IHC staining of NB samples were performed to detect its effects on the protein levels of TF.The protein levels of TF in Ganglia tumor,well differentiated,poorly differentiated and undifferentiated NB tissues were examined to determine their potential clinical significance.Results: To determine the interacting partner of RBPJ,we analyzed the publicly available datasets Bio GRID,IID and STRING.Based on overlap analysis,seven proteins that theoretically interact with RBPJ.To further narrow down the interacting partner of RBPJ,we used IP and Coomassie blue staining,we identified a protein band between 55 and 70 KD.These results indicate that of the seven potential binding partners,SNW1 may interact with RBPJ.RBPJ/SNW1 antibody pull-down assays and Bi FC assay indicated that SNW1 and RBPJ interact with each other.Mining of publicly available microarray data(GSE62564)showed that SNW1 is highly expressed in NB with progression(P < 0.001,unpaired t-test),INSS stages 2 vs.4(P < 0.001,unpaired t-test),MYCN amplification(P < 0.001,unpaired t-test),INSS stages 1 vs.4(P < 0.001,unpaired t test).Notably,SNW1 as an independent marker of NB progression and outcome and positively correlated with RBPJ in public databases(R?=?0.3905,P?<?0.001).Consistently,IHC staining of NB samples revealed higher SNW1 expression in non-differentiated tissue than in differentiated tissue.Conclusions: These data demonstrated that SNW1 interact with RBPJ in NB.SNW1 as an independent marker of NB progression and outcome and positively correlated with RBPJ Part three: SNW1 acts as a negative regulator of Notch target genes via RBPJObjective: To investigate the possible role of SNW1 in the Notch signaling of neuroblastoma(NB).Methods: Depletion of RBPJ use two independent short hairpin RNA(sh RNA),sh-RBPJ#1 and RBPJ#2,in NB cell lines.The q RT-PCR assays were applied to observe the transcription levels of Notch signaling target gene via stable over-expression and knockdown of SNW1 on NB cell lines.SNW1 and RBPJ rescue experiments was done to clarify that SNW1 how to regulate Notch signaling pathway target genes.Results: The q RT-PCR assays indicated that stable transfection of SNW1 leads to down-regulation of HES1,HES4,HEY1,and HEY2.Depletion of SNW1,using two independent short hairpin RNAs(sh RNAs)sh-SNW1 #1 and sh-SNW2 #2,leads to upregulation of HES1,HES4,HEY1,and HEY2.However,while knockdown of RBPJ can prevent SNW1 regulate Notch target genes in NB cells with stable transfected with empty vector(mock),SNW1,scramble sh RNA(sh-Scb),or sh-RBPJ#1.Conclusions: SNW1 contribute to the suppression of Notch target genes in NB cell lines via interacting with RBPJ.