| ObjectiveHerein,this article aims to study the effects of SAHA(a histone deacetylase inhibitor)on neuroblastoma killing and its synergistic effect with MG132(a proteasome inhibitor).Besides,we explore the molecular mechanism of SAHA on neuroblastoma inhibiting separately.MethodsHuman neuroblastoma cell line SH-SY5Y?SK-N-AS,NGP and SK-N-BE2 were cultured.The Cell counting Kit 8 and RTCA real time cellular analysis were used to detecte Cell proliferation in the groups which exposured with SAHA,MG132,the combined of the two and the overexpression of ING5.The Seahorse XF Extracellular Flux Analyzer was used to measured mitochondrial respiration and glycolysis.The Flow cytometry was used to measured the cell opoptosis assay and Cell cycle analysis.Cell migration and invasion ability were measured using Wound healing and transwell assays.qRT-PCR and Western blot analysis were used to investigate the related expression of mRNA and proteins.Chromatin immunoprecipitation(ChIP)was used to detect combination of the AC-H3?AC-H4 or ING5 proteins with the promoter of the target genes.Co-immunoprecipitation(Co-IP)was used to detect the protein interaction of AC-H3?AC-H4 and ING5.The co-immunoprecipitation of double ChIP was used to detect the combination of the proteins of AC-H3 and AC-H4,AC-H3 and ING5,with the promoter of the target genes.Biological information database was used to calculate the micoRNA which can target the ING5 gene,then used the qRT-PCR to detect the expression of miR-640,miR-543,miR-624-3p and miR-196-b after SAHA exposure.In addition,Western blot analysis were used to investigate the related expression of ING5 protein after transfected the mimics and inhibitor of the micoRNA.Effect of SAHA?MG132?SAHA combine MG132 exposured and ING5 overexpression in vivo was determined by subcutaneous and orthotopic xenografts in nude mice.Immunohistochemistry and Western blot assay were used to investigate the related expression of the proteins of AC-H3?AC-H4?ING5?PTEN,p53,Ki-67,Lc-3b in the nude tumors,TUNEL staining was were used to detect the apoptosis.Pathology and tissue microarray(TMA)analysis and Immunohistochemistry were used to investigate the related expression of the proteins of AC-H3?AC-H4 and ING5 in the tissues of human neuroblastoma,The clinicopathological and pathological staging value were evaluated for neuroblastoma samples according to TNM staging system and World Health Organization(WHO)classification system.Results1.SAHA and MG132 reduced proliferation of SH-SY5Y cells in both a time-and dose-dependent manners.SAHA combine MG132 exposured in small concentration has synergistic effect(P<0.05).2.SAHA and MG132 exposure apparently inhibited the energy metabolism,induced Gi arrest,promoted apoptosis in a concentration-dependent manner,and decreased cell migration and invasion in a concentration-dependent manner(P<0.05).3.SAHA and/or MG132 treatment up-regulated the expression of ING5,PTEN,p53,Caspase-3,Bax,p21 and p27,while down-regulated the expression of 14-3-3,MMP-2,MMP-9,ADFP,Nanog,c-myc,CyclinD1,CyclinB1 and Cdc25c(P<0.05).SAHA treatment enhanced the acetylation of histones H3 and H4,however this effect was not seen with MG132 exposure.4.ING5 overexpression had the same inhibiting effects on the malignant biological behavior of neuroblastoma as SAHA expose(p<0.05).The expression of phenotype-related proteins was the same in ING5 transfectants as in SAHA-treated SH-SY5Y cells(P<0.05).5.Acetyl-histone H3,acetyl-histone H4 and ING5 were demonstrated to bind to the special fragment of p21,p27,c-myc,cyclinD1 and nanog promoters.SAHA treatment and ING5 overexpression may be involved in recruiting more Ac-histone H3,Ac-histone H4 and ING5 proteins to these promoters.The interaction between Ac-histone H3,Ac-histone H4 and ING5 was strengthened by SAHA exposure and ING5 overexpression.6.SAHA treatment and ING5 overexpression decreased mRNA expression of c-myc,Nanog and CyclinD1,but increased mRNA expression of p27 and p21(P<0.05).7.There is a combine between miR-640,-543,-624-3p,-196-b and the mRNA of ING5.Only miR-543 and miR-196-b overexpression decreased ING5 protein expression in SH-SY5Y cells,while SAHA might ameliorate inhibition.The converse was also true for their inhibitor.8.SAHA exposure significantly inhibited viability in the cell lines of SK-N-AS,NGP and SK-N-BE2 in both concentration-and time-dependent manners(p<0.05).SAHA treatment caused the down-regulated expression of miR-543 and-196-b in these cell lines to increase the expression of ING5.Overexpression of ING5,Ac-H3,Ac-H4,p21 and p27,and the underexpression of CyclinD1,CyclinB1,c-myc and Nanog were observed in the three cell lines after exposure to SAHA.9.Tumor volumes of SH-SY5Y xenografts were smaller than control when treat with SAHA or MG132 alone,while the synergistic effect was observed when treatments were combined.ING5 overexpression had the same results as SAHA treatment(P<0.05).In both SAHA treatment and ING5 overexpression conditions,neuroblastoma cells had a higher expression of acetyl-histones H3 and H4 and p53 than control.Ki-67 immunostaining and TUNEL assay demonstrated that the inhibitory effect has a positive correlation with a low proliferation and high apoptosis in compare with control.10.Acetylated histones H3 and H4 were predominant expressed in the nuclei of neuroblastoma cells,while ING5 was found in the cytoplasm or nucleus.The Ac-histone H3 or cytoplasmic ING5 expression was positively linked to neuroblastoma tumor size(P<0.05).Nuclear ING5 expression was negatively linked to neuroblastoma tumor size(P<0.05).Conclusions1.In vivo and vitro experiments,SAHA and MG132 exposed and ING5 overexpression reduced proliferation,inhibited the energy metabolism,induced cell cycle arrest,promoted apoptosis,decreased cell migration and invasion to has its anti-neuroblastoma effects.2.SAHA exposure could decrease the miR-543 and miR-196-b indirectly which resulted in ING5 overexpression in neuroblastoma cells.3.SAHA exposure could increase the expression of ING5 and promote the expression of AC-H3 and AC-H4 by inhibiting the histone deacetylase.4.AC-H3,AC-H4 and ING5 proteins had a binding interaction,bind to the special fragment of p21,p27,c-myc,cyclinD1 and nanog promoters.5.The expression of these acetyl-histones and ING5 may thus be involved in neuroblastoma tumor growth. |
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