Long Non-coding RNA MZF1-AS1 Through Facilitating Interaction Of PARP1 With E2F1 To Regulate Neuroblastoma Progression And Its Underlying Mechanism

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Long non-coding RNA MZF1-AS1 is associated with tumor progression in neuroblastomaObjective: To investigate long non-coding RNAs(lncRNAs)associated with human neuroblastoma progression,and to evaluate the effects of MZF1-AS1 on neuroblastoma and its expression in neuroblastoma.Methods: Comprehensive analysis of the publicly available data(GSE16476 and GSE62564)derived from Gene Expression Omnibus(GEO)database,and evaluated the prognostic significance of lnc RNA in NB patients.Real-time quantitative PCR and Northern-blot assays were performed to detect the expression of MZF1-AS1 in NB cell lines.And,RNA FISH was used to investigate the localization of MZF1-AS1 in cells.According to the expression of MZF1-AS1 in NB cell lines,cells with MZF1-AS1 overexpression and knockdown stably transfected were established.Then,MTT,soft assay for colony formation and matrigel invasion assays were performed to evaluate the effect of MZF1-AS1 on proliferation and invasion of NB in vitro.In addition,subcutaneous xenografts tumors and tail vein metastasis assays in nude mice were utilized to assess the effect of MZF1-AS1 on proliferation,invasion and metastasis of tumor in vivo.Results: Comprehensive analysis revealed that MZF1-AS1 was a novel lnc RNA,which was closely associated with tumor progression in neuroblastoma.The result of RNA FISH experiment demonstrated that MZF1-AS1 located in the nucleus.Meanwhile,MZF-AS1 overexpression facilitated the anchorage-independent growth and invasion in neuroblastom cells,which was repressed by knocking down MZF1-AS1.For the in vivo assays,ectopic expression of MZF-AS1 resulted in increased tumor volume,tumor weight and metastasis and stable silencing of MZF1-AS1 reduced tumor volume,weight and metastasis.Conclusions: MZF1-AS1 is closely associated with tumor progression in neuroblastoma,which can promote the proliferation,invasion and metastasis of neuroblastoma cells in vitro and in vivo.Part ? Long non-coding RNA MZF1-AS1 interacts with PARP1 in neuroblastomaObjective: To investigate MZF1-AS1 associated protein and elucidate the mechanism of interaction between MZF1-AS1 and PARP1.Methods: The proteins that bounded to MZF1-AS1 were analyzed by RNA pull-down assay and mass spectrometry.And,cat RAPID was introduced to evaluate the binding propensity of protein and MZF1-AS1.RIP,RNA EMSA and in vitro binding assays were performed to investigate the cooperation between MZF-AS1 and PARP1,and associated mechanism.Results: The results of RNA pull-down assay and mass spectrometry revealed that the nuclear protein PARP1 was a partner of MZF-AS1.Cat RAPID algorithm analysis uncovered a high possibility of binding between MZF-AS1 and PARP1,and analyzed the structural basis of their interaction.RIP assay further proved theem interaction.Moreover,RNA EMSA indicated that the exon 6 of MZF1-AS1(1240-1639 bp)could bind with PARP1.And,in vitro binding assays using PARP1 recombinant protein demonstraed that the BRCT domain of PARP1(376-662 amino acids)mediated their interaction.Conclusions: Long non-coding RNA MZF1-AS1 interacts with PARP1 protein in neuroblastoma.Part ? PARP1 cooperates with Long non-coding RNA MZF1-AS1 to regulate genes expressionObjective: To clarify the role of PARP1 in the expression of MZF1-AS1 regulated genes.Methods: RNA-seq assay was introduced to investigate long non-coding MZF1-AS1 regulated genes.Bio GRID and Ch IP-X were used to analysis corresponding transcript factors and target genes.The roles of PARP1 on MZF1-AS1 regulated gene expression were evaluated by dual-luciferase,Ch IP,qRT-PCR and western blot assays.Results: The results of RNA-seq revealed that MZF-AS1 could regulate genes expression.Comprehensive analysis of Bio GRID and Ch IP-X demonstrated that E2F1 was the most important transcript factor that regulated genes expression and interacted with PAPR1.Furthermore,dual-luciferase,Ch IP,q RT-PCR and western blot assays indicated that MZF1-AS1 and PARP1 coordinate regulated gene expression.Conclusions: PARP1 cooperates with MZF1-AS1 to regulate genes expression.Part ? Long non-coding RNA MZF1-AS1 facilitates the interaction between PARP1 and E2F1Objective: To elucidate the interaction of PARP1 with E2F1,and to explore the effect of MZF-AS1 on them interaction.Methods: Co-IP and Bi FC assays were used to detect PARP1 and E2F1 interaction.And,different truncations of PARP1 and E2F1 were constructed to investigate the mechanism of them interaction,and to evaluate the interaction under different conditions.Ch IP,q RT-PCR and western blot assays were performed to evaluate the effects of PARP1 and E2F1 interaction on genes expression.Additonally,soft agar colony formation and matrigel invasion assays were utilized to evaluate the effect of MZF1-AS1,PARP1 and E2F1 interaction on proliferation and invasion of neuroblastoma cells in vitro.Results: The result of co-IP and BiFC assays demonstrated that PARP1 interacted with E2F1 directly.And,co-IP assay revealed that the BRCT domain of PARP1 and DNA binding domain(DBD)of E2F1 were responsibled for them interaction.Meanwhile,MZF-AS1 enhanced PARP1 and E2F1 interaction without affecting the poly(ADP-ribosyl)ation levels of E2F1.Ch IP and q PCR,q RT-PCR and western blot assays demonstrated that the interaction of PARP1 and E2F1 affected genes expression.For the in vitro assays,the results of soft agar colony formation and matrigel invasion assays indicated that MZF1-AS1 affected the proliferation and invasion of neuroblastoma cells,which depended on PARP1 and E2F1.Conclusions: MZF1-AS1 regulates genes expression by facilitating the interaction between PARP1 and E2F1.Part ? PIP-14 inhibits the interaction between MZF1-AS1 and PARP1Objective: To investigate the effect of PIP-14 on the interaction of MZF1-AS1 and PARP1,and to assess the effect of PIP-14 on neuroblastoma progression.Methods: RNABind RPlus,a platform that predicted RNA-binding residues in proteins was introduced to analyze the binding site of nucleic acid and protein interaction.RNA pull-down assay was used to analysis the binding between PARP1 mutant and MZF1-AS1.And,immunofluorescence assay was utilized to observe PIP-14 uptake and cellular localization.The effects of PIP-14 on MZF1-AS1 and PARP1 interaction were investigated by RIP and RNA pull-down assays.Western blot assay was applied to detect the effect of PIP-14 on downstream genes expression.The effects of PIP-14 on proliferation and invasion of neuroblastoma cells in vitro were determined by soft agar colony formation and matrigel invasion assays.In addition,subcutaneous xenografts tumors and tail vein metastasis assays in nude mice were utilized to evaluate the effects of PIP-14 on proliferation,invasion and metastasis of tumor in vivo.Results: RNABind RPlus analysis demonstrated that three amino acids in WGR region of PARP1 were the major sites responsibled for PARP1 binding with MZF-AS1.Then,RNA pull-down assay showed that the mutation of binding sites inhibited nucleic acid and protein interaction.Immunofluorescence assay revealed that PIP-14 could be taken in by tumor cells and mainly distributed in the nucleus.And,RIP and RNA pull-down assay suggested that PIP-14 could inhibit nucleic acid-protein interaction,and western blot assays demonstrated that PIP-14 repressed downstream genes expression.For the in vitro assays,administration of PIP-14 led to suppress the proliferation and invasion of neuroblastoma cells.In addition,compared to the control group,nude mice received the PIP-14 treatment had smaller tumor volume,lower weight and less metastasis in vivo assays.Conclusions: PIP-14 inhibits the interaction between MZF1-AS1 and PARP1,and suppresses neuroblastoma progression.Part ? Analysis of genes expression in neuroblastomaObjective: To detect the expression of genes in neuroblastoma clinical specimens and cell lines,and to evaluate the correlation between gene expression and survival.Methods: q RT-PCR and western blot assays were performed to evaluate the gene expression of PARP1,E2F1,MZF1,c-Kit,PRKCG and RET in different neuroblastoma tissues and cell lines.The GEO database was utilized to analyze the correlation of PARP1,E2F1,MZF1,c-Kit,PRKCG and RET gene expression with survival.Results: The results of q RT-PCR and western blot assays suggested that the expression of PARP1,E2F1,MZF1,c-Kit,PRKCG or RET was higher in different NB tissues and cell lines than normal tissues.In addition,Kaplan–Meier survival analysis derived from GEO database revealed that NB patients with high PARP1,E2F1,MZF1,c-Kit,PRKCG or RET expression had poor prognosis.Conclusions: The expression of PARP1,E2F1,MZF1,c-Kit,PRKCG or RET is higher in different neuroblastoma tissues and cell lines than normal tissues,and NB patients with high expression of PARP1,E2F1,MZF1,c-Kit,PRKCG or RET have worse overall survival than those with low expression.