The Role Of TSPYL5 On Maintaining The Characters Of CSCs In Neuroblastoma Via P53 Signaling Pathway

Objective:Testis-specific-like protein 5(TSPYL5),a member of TSPY-TSPYL-SET-NAP1 super-family,has a highly conserved domain of nucleosome assembly protein 1(NAP1)at its C-terminal.NAP1 is a histone chaperon and plays an important role in chromatin assembly,tissue-specific transcription regulation,histone shuttling and cell-cycle regulation.Present studies reveal that TSPYL5 is involved in many tumors,human pluripotent stem cells(hPSCs),angiogenesis and spermatogenesis.The knock-down of TSPYL5 down-regulated the expression of differentiation-related genes and up-regulated that of pluripotent genes in hPSCs,which suggested it was involved in the function of stem cells.However,the roles of TSPYL5 in cancer stem cells(CSCs)remains unclear.In our previous work,we discovered TSPYL5 expressed highly in mouse brain and testis,which was consistent with the high expression of TSPYL5mRNA in the nervous system and male reproductive system in the human protein atlas database.And the database showed that TSPYL5 mRNA was also highly expressed in the neuroblastoma cell line SH-SY5Y.Neuroblastoma,an embryonic cancer,derives from aberrant neural crest(NC)cells.NC cells are pluripotent,similar to stem cells.Consideration that the stem cells and cancer stem cells share some common biological phenotypes and molecular signal pathways,and CSCs ratio is reported as high as 0.8%-51%in NB,therefore,our study applies neuroblastoma cell lines as a cell model to research the role of TSPYL5 in CSCs.Materials and Methods:Part Ⅰ:Neuroblastoma cell lines were screened as cellular study model.Lenti-viral shRNA vectors targeting TSPYL5(shTSPYL5)or negative control(shNC)were transfected into SK-N-SH cells and SH-SY5Y cells.Rescue cells were constructed using lenti-viral expression vector that encoded degenerate bases of TSPYL5 transfecting into shTSPYL5 cells,and Rescue cells were applied for function recovery experiments.Stable expression of shTSPYL5,shNC or Rescue cells were applied to detect sub-population cells by flow cytometry,to test drug sensitivity by CCK-8,to determine migration and invasion by scratching and transwell test,to evaluate stemness properties by sphere formation test and to assess tumorigenity by xenograft assay.Survival analysis was done between the NB patients with high level of TSPYL5 and low level of TSPYL5 in the cBioportal data set.Part Ⅱ:RNA sequencing assay(RNA-seq)was applied to analyze the effect of TSPYL5 knocked-down on the transcriptome of SK-N-SH cells.RT-qPCR was used to validate the differentially expressed genes of NB CSCs markers between the shTSPYL5 and shNC of SK-N-SH or SH-SY5Y cells.Based on the cBioportal data set,correlation analysis was done between the TSPYL5 mRNA and the CSCs markers in NB patients.GO and KEGG enrichment pathways were analyzed for seeking TSPYL5-related biological signal pathway and cellular function.RT-qPCR was validated the transcription expression of major p53-target genes.Correlation analysis was done between the TSPYL5 mRNA and the p53-target genes in NB patients from cBioportal data set.Part III:Sanger sequencing was applied to identify the genotype of p53.RT-qPCR test the transcription level of p53 in shTSPYL5 cells and shNC cells.SK-N-SH and SH-SY5Y cells over-expressing TSPYL5(o.v.TSPYL5)were constructed.The effects of TSPYL5 on the subcellular localization of p53 were analyzed by immunofluorescence and cytoplasm-nucleus isolation assay.IP-MS was used to screen the potential proteins interaction with TSPYL5 by using TSPYL5 antibody as a bait.The interaction between TSPYL5 and G3BP1 was verified by in vitro transcription and translation combined with co-IP experiment.The interaction between TSPYL5 and USP10 was investigated by in vitro transcription and translation combined with co-IP experiment.Structure domain co-IP assay and molecular docking mimics were done to identify the interaction domain between TSPYL5 and G3BP1 or USP10.Two-round IP was applied to determine whether TSPYL5,G3BP1 and USP10 to form a complex.IP assay was applied to confirm the impact of TSPYL5 on the interaction with G3BP1 and USP10.The effect of TSPYL5 on p53 ubiquitination was detected by Western blot.USP10 was knocked down by siRNA to detect whether the effect of TSPYL5 on p53 ubiquitination was dependent on USP 10.Results:Part Ⅰ:SK-N-SH cells and SH-SY5Y cells stably expressing shTSPYL5,shNC or Rescue were successfully established identified by western blot.Compared with shNC cells,shTSPYL5 cells demonstrated decreased rate of sub-population cells,more sensitive to cisplatin,less ability of migration and invasion,less and smaller tumor spheres.SK-N-SH shTSPYL5 cells showed failure tumorigenesis in the xenograft model.Function recovery experiments with Rescue cells showed the sub-population rate,drug sensitivity,migration,invasion and tumor sphere formation rescued the function of shTSPYL5 cells,which suggested TSPYL5 knocked-down had no off-target effect.The K-M plot survival analysis of NB patients in the cBioportal database showed that there was no significant statistical difference between patients with high level of TSPYL5 and low level of TSPYL5.But,we found that the survival rate of patients with low level of TSPYL5 is significantly higher than that patients with high level of TSPYL5 by making the survival time more than 40 months as subgroup analysis(p<0.01).Part Ⅱ:Transcriptome sequencing illustrated that TSPYL5 knockdown had a significant effect on gene expression in SK-N-SH cells.And a total of 14697 differentially expressed genes(DEGs)were detected,of which 7,262 genes were significantly up-regulated and 7,435 genes were significantly down-regulated.RT-qPCR was applied to validate the CSCs markers which were LMNA,L1CAM,DLK1,CD24,CD44,LGR5 and Fzd6 in SK-N-SH cells,and the expression of these genes excluded Fzd6 suggested CSCs biological function was down-regulated by TSPYL5 knocked-down.Targeted detection of the above genes in SH-SY5Y cells showed that the expression of LMNA,L1CAM,DLK1 and LGR5 were also significantly different between shTSPYL5 and shNC cells,suggesting that knocked-down TSPYL5 down-regulated CSCs.The analysis of cBioportal database suggested that the expression level of TSPYL5 in NB patients was correlated with the expression levels of marker molecules CD24,LMNA and L1CAM of CSCs,and was negatively correlated with the change of CSCs function indicated by the three markers.Go and KEGG enrichment analysis showed 59 DEGs in p53 signal pathway.RT-qPCR verified the expression changes of PAI,DRAM,p53R2,p21and GADD45A,which were the main downstream target genes of p53.The trend suggested that TSPYL5 knocked-down enhanced the function of p53 as a transcription factor.The analysis of cBioportal database data showed that the expression of TSPYL5 in NB patients was negatively correlated with the transcription of p53 downstream target genes PAI,DRAM,p21 and GADD45A.These evidences suggested knocked-down TSPYL5 increased the function of p53.Part III:Genotype identification showed that p53 was wild type in SK-N-SH and SH-SY5Y cells.RT-qPCR showed that there was no significant difference in p53 expression between shTSPYL5 and shNC cells.Under non-stress or stress states(X radiation),after MG132 treated cells,immunofluorescence and nucleoplasm isolation experiments showed that p53 was significantly sequestered to cytoplasm in o.v.TSPYL5 and Ctrl cells,while p53 was only localized in nucleus in shTSPYL5 cells.A total of 85 protein peptides were identified by IP-MS using TSPYL5 as bait protein,and the abundance of G3BP1 was 238461.953125.The co-IP experiment verified the interaction between TSPYL5 and G3BP1 in cells.In vitro protein expression system combined with co-IP experiment further confirmed the direct physical binding between TSPYL5 and G3BP1.The expression of USP10 was increased in cells under stress.The co-IP assay suggested that USP10 interacted with TSPYL5 in cells.In vitro protein expression system combined with co-IP experiment further confirmed the direct physical binding between USP10 and TSPYL5.The two-round IP test showed that G3BP1-TSPYL5-USP10 formed a trimolecular complex.The domain co-IP experiment suggested that TSPYL5 could bind to the domains of G3BP1 aa200-460 and USP10 protein aa400-797,respectively.The molecular docking mimics further supported the experimental results of domain co-IP by computer modeling.IP assays showed that TSPYL5 enhanced the binding ability of G3BP1 to USP10,which competitively suppressed the binding of USP10 to p53,thereby,the deubiquitination of p53 and mediating p53 into the nucleus by USP10 was inhibited.Ubiquitination assay further confirmed that knockdown of TSPYL5 reduced p53 ubiquitination and over-expression of TSPYL5 increased p53 ubiquitination.The role of TSPYL5 on regulating deubiquitination of p53 was counteracted by knocked-down of USP10 with siUSP10,suggesting that TSPYL5 was dependent on USP10 to regulate p53 deubiquitination modification.Summary:1.The significant impact of TSPYL5 on cell phenotypes and the expression of CSCs markers,suggested TSPYL5 was an important molecular in maintaining the characters of CSCs.2.TSPYL5 obviously suppressed the transcriptional role of p53,which could be one of causes to maintaining CSCs by regulating the expression of CSCs markers.3.TSPYL5 interacted with G3BP1 and USP10,respectively,to form a trimolecular complex.Among them,TSPYL5 competitively suppressed the binding of p53 to USP10 by strengthening the binding of G3BP1 and USP10,which down-regulated the deubiquitination of p53 and significantly reduced the nuclear location of p53,so that it suppressed the role of p53 as a transcription factor.3.TSPYL5 occupied the catalytic core domain of USP10 aa400-792,which could down-regulate the deubiquitination of p53 by suppressing USP10 and regulate the cytoplasmic-nuclear location of p53.Conclusion:In this study,the phenomenon that TSPYL5 regulates the nuclear-cytoplasmic subcellular localization of p53 is first discovered,and its molecular mechanism is explained.In the cytoplasm,TSPYL5 forms a complex with G3BP1 and USP10,which strengthens the binding ability of G3BP1 and USP10,to competitively suppress the binding of p53 and USP10,and down-regulates the deubiquitination of p53.The ’p53 ubiquitination/deubiquitination-p53 cytoplasmic/nuclear shuttling’system in cells is one of the core mechanisms regulating the function of p53,which has attracted the attention of researchers.Previous studies have reported that TSPYL5 competed with p53 and combined with USP7 to suppress p53 deubiquitination.In this study,we found another important deubiquitinating enzyme USP10 mediated TSPYL5-related p53 pathway,and further outlined the important role of TSPYL5 in regulating p53 function.The expression of TSPYL5 is highly tissue specific,and it is only expressed in testis and nervous system under physiological conditions.Therefore,in TSPYL5-expressing and p53 wild-type tumors,targeted suppressing the function of TSPYL5 may not have a significant impact on the normal physiological function,and can specifically active the function of p53 to play an anti-tumor role.Therefore,our findings may provide new clues for the treatment of some tumors including NB.