| PartⅠPrognostic value of m6A methylation regulators on neuroblastoma in childrenObject:m6A RNA methylation is closely related to the occurrence and development of tumors.This part aims to explore the prognostic effects of m6A methylation regulators on pediatric neuroblastoma.Methods:RNA sequencing data and clinical characteristics of 498 neuroblastoma patients were obtained from the comprehensive gene expression database.We applied bioinformatic methods to investigate the expression of 20 published m6A methylation regulators.R package of limma was used to analyse the differencial expression in each clinical characteristic.Based on m6A regulators,unsupervised gene consensus clustering analysis was used to conduct unsupervised clustering for all samples.Kaplan-meier method was used to analyse the overall survival rates.The biological functions of different m6A modification modes were analyzed by GSVA.Univariate Cox,LASSO and multivariate Cox regression analysis were used to establish the prognostic risk model.The accuracy of the prognostic models was evaluated by time-dependent receiver operating characteristic（ROC）curve.Considering the median risk score as the cutoff,the patients were divided into high-and low-risk group.Kaplan-Meier method was used to analyze the overall survival curve of the high-and low-risk group.Univariate and multivariate Cox regression analyses were performed to observe the influence of m6A prognostic model and other clinical features on overall survival.Subgroup analysis was used to evaluate the robustness of the model in subgroup of different pathological features.The robustness of the model was further verified using the children’s Tumor Information Database（TARGET）data as an external data set.GSEA was used to explore the potential biological function of the m6A prognostic risk model.Results:（1）m6A regulators were differencially expressed between subgroups stratified by progression,COG risk and INSS stage in neuroblastoma,which were not caused by gene mutations and copy number variation,but at the transcriptome level.（2）The overall sample was defined as group A,B and C by unsupervised clustering based on m6A regulators.Group B had the highest degree of malignancy and the lowest overall survival rate.Group A had the lowest degree of malignancy and high overall survival rate.Group C was in the middle.According to GSVA functional enrichment analysis,group B and C was more associated with malignant biologic features of tumors,including DNA repair,oxidative phosphorylation,glycolytic metabolism,cell apoptosis inhibition,PI3K-Akt-MTOR pathway,activation of E2F and MYC transcription factor.（3）The risk model consists of METTL14 HNRNPC YTHDF1 WTAP and IGF2BP2.（4）The ROC curve showed that the model had high sensitivity and specificity.The results of subgroup analysis showed that the risk score differed significantly among subgroups in different pathological characteristics.Multivariate Cox analysis showed that the risk model was an independent prognostic index in predicting the overall survival of children with neuroblastoma.（5）GSEA results showed that high-risk group was associated with mTORC1 signal transduction pathway,MYC transcriptional activation,E2F transcriptional activation and DNA repair.Conclusion:（1）m6A methylation regulators were associated with clinicopathological features and overall survival prognosis of children with neuroblastoma.（2）The survival prognosis model based on five m6A methylation regulators（METTL14HNRNPC YTHDF1 WTAP and IGF2BP2）can accurately predict the overall survival rate of children with neuroblastoma.PartⅡClinical significance of YTHDF1 in neuroblastomaObject:This part aim to further explore the prognostic effect of YTHDF1 on neuroblastoma and predict the underlying mechanism.Methods:The expression values of YTHDF1 and clinical information of 498 patients in GEO database were statistically analyzed.The relationship between YTHDF1 and clinical features of the children was evaluated by Chi-square,test,Wilcoxon test and kruskal-Wails H test.Kaplan-Meier method and subgroup analysis were used to evaluate the overall survival rate of children with high and low expression of YTHDF1.Univariate and multivariate Cox regression analysis were used to determine the independent effect of YTHDF1 on survival prognosis.For functional enrichment analysis,GO and KEGG were used to screen cell functions and signaling pathways that were significantly enriched with high expression of YTHDF1 genes.Me-RIP-SEQ and e Clip-seq data were downloaded from m6A2Target online database,and potential targets of YTHDF1 were screened with RNA-seq data and STRING database.results:（1）There were significant differences of YTHDF1 expression levels among the subgroups stratified by age,MYCN amplification status,COG risk,Histology feature,and tumor progression.（2）There was a significant difference in overall survival between the group with high and low YTHDF1 expression（Logrank test P<0.0001）.The 5-year survival rate of the low expression group was 88.4%（95%CI:84.4-92.6%）,while the high expression group was69.1%（95%CI:63.4-75.4%）.Multivariate Cox regression analysis showed that high YTHDF1 expression was still associated with lower overall survival after adjustment for multiple clinical covariates（HR=2.32,95%CI:1.22-4.43）.（3）YTHDF1 binding MYC,CTNNB1,DYNC1H1,BARD1,BDNF,GSG2,CXCR4,JUN and LIG4 were YTHDF1 binding Candidate genes.Further analysis showed that MYC was more likely to be the target gene.Conclusion:（1）YTHDF1 showed differential expressed in each pathological features,including age,MYCN amplification status,Histology,and tumor progression.（2）The overall survival rate of patients with high and low expression of YTHDF1 is significantly different,and YTHDF1 has an independent effect on the prognosis.（3）MYC may be a potential target gene for YTHDF1 binding.PartⅢ Effect and mechanism of YTHDF1 on neuroblastomaObject:This part aims to verify the effects of YTHDF1 on the malignant biology of neuroblastoma cells and the regulation of MYC.Methods:We collected twelve first diagnosed neuroblastoma pathological specimens in the first hospital of Jilin university in the past two year to detect the expression level of YTHDF1.SK-N-FI and IMR-32 cells were used on the subsequent experiments:（1）mRNA expression level was detected by q RT-PCR;（2）Western Blot was used to detect protein expression level;（3）CCK-8 and cell cloning assay were used to detect cell proliferation;（4）Transwell chamber was used to detect the migration and invasion of cells.（5）The m6A modification level of MYC mRNA was detected by Me-RIP-q PCR.（6）RIP-q PCR was used to detect the binding ability of YTHDF1 and MYC mRNA.Results:（1）The mRNA and protein expression levels of YTHDF1 in high stage tumor tissues were significantly higher than that in low stage.（2）The results of CCK-8 results showed that the cell activity of the YTHDF1knockdown group was significantly decreased,while that of the YTHDF1 overexpression group was significantly increased.The results of cell cloning experiment showed that the cell cloning efficiency of YTHDF1 knockout group was significantly reduced,while that of YTHDF1 overexpression group was significantly increased.（3）The results of migration and invasion experiments showed that the number of cells crossing the chamber was significantly reduced in the YTHDF1 knockdown group,while the number of cells crossing the chamber was significantly increased in the YTHDF1overexpression group.（4）In the YTHDF1 knockdown group,the expression level of MYC protein was decreased and RNA level was not significantly changed,while in the YTHDF1overexpression group,the expression level of MYC protein was increased and RNA level was not significantly changed.RIP-q PCR results showed that MYC gene mRNA was enriched in the YTHDF1-binding RNAs,and the addition of si-METTL3 and methylation inhibitor 3-Deazaadenosine could reverse the increase of MYC protein induced by overexpression of YTHDF1.（5）Me-RIP-qPCR results showed that MYC mRNA was significantly enriched.（6）After MYC knockdown,the proliferation,migration and invasion ability of SK-N-FI cells were decreased,and apoptotic cells increased.（7）The effect of YTHDF1 overexpression on biological behavior of SK-N-FI cells could be partially reversed after MYC knockdown.Conclusion:（1）YTHDF1 promoted proliferation,migration and invasion of neuroblastoma cells.（2）YTHDF1 regulated MYC protein expression in an m6A dependent manner.（3）YTHDF1 promoted proliferation,migration,invasion of neuroblastoma cells through MYC.PartⅣEffect and mechanism of YTHDF1 on neuroblastomaObject:We aimed to preliminarily explore the effect of 20（S）-propanaxadiol（PPD）on malignant biological behavior of neuroblastoma.Methods:SK-N-FI cell was used as research object for experiments:（1）CCK-8 and cell cloning assay were used to detect cell proliferation;（2）Transwell chamber was used to detect the migration and invasion of cells.（3）Western Blot was applied for detecting protein levels;（4）The overall m6A level was detected by colorimetry.Results:（1）Results of CCK-8 showed that 20（S）-PPD significantly inhibited the proliferation of SK-N-FI cells in a dose-dependent manner,with IC50 value of 34.35μM at 24h.The results of cell cloning and formation experiment showed that the proliferation ability of 20（S）-PPD cells was significantly reduced,and the proliferation ability of SK-N-FI decreased with the increase of 20（S）-PPD concentration.（2）Results of Transwell chamber experiment showed that the number of SK-N-FI cells passing through the chamber decreased with the increase of 20（S）-PPD concentration.（3）After SK-N-FI cells were treated with 20（S）-PPD,YTHDF1 did not show significant change.However,a surprising change of the total m6A level has taken place.（4）After 20（S）-PPD treatment,METTL3 did not change significantly,and MYC protein level decreased.Overexpression of METTL3 could rescue the change of binding ability between MYC mRNA and YTHDF1 protein caused by 20（S）-PPD treatment.Conclusion:（1）20（S）-PPD inhibited the proliferation,migration and invasion of neuroblastoma SK-N-FI cells.（2）20（S）-PPD decreased the m6A level of neuroblastoma SK-N-FI cells,and by which to down-regulate MYC protein level. |
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