| Background:Medulloblastoma(MB)is the most common brain tumor of childhood,with an overall annual incidence of approximately 5 cases per million in the pediatric population.Due to the high degree malignancy of MB,the overall survival rate is still only about 70%even after standard treatment(surgical resection,radiotherapy and adjuvant chemotherapy).MB is mainly divided into four subgroups in clinic:WNT,SHH,Group 3 and Group 4,and the molecular characteristics among these subgroups are significantly different.Matching molecular groups to appropriate therapies might contribute to improve survival and reduce morbidity associated with long-term treatment.The current understanding of the molecular mechanisms of different subgroup of MB is mainly focused on protein-coding genes,while the understanding of the role of lncRNA in the molecular pathogenesis of MB is limited.Accumulating data suggest that lncRNA has been found to participate in the genesis and development of tumors through a variety of mechanisms in other tumors,a systematic and in-depth study of characteristics of lncRNA expression and regulation in MB will advance in the understanding of the biology and molecular pathogenesis of MB.Methods:In this study,we first performed rRNA-depletion paired-end 150 nt strand-specific RNA-seq on 63 MB tumor tissue samples with NovaSeq sequencing platform.Secondly,the overall expression characteristics of lncRNA in MB were explored through methods including lncRNA and circular RNA(circRNA)ab initio annotation and functional enrichment analysis.Thirdly,we used mRNA,lncRNA and circRNA expression data to classify MB,and compared the performance of different classification methods,so as to determine the final subgrouping strategy.Then,the subgroup-specific lncRNAs were identified by differential expression analysis.Next,through functional enrichment analysis and gene co-expression network,we explored the regulatory roles of subgroup-specific lncRNAs in each subgroup of MB.Finally,DNA methylation sequencing was performed on 16 MB samples,and the DNA methylation regulation characteristics of lncRNA and mRNA genes were systematically compared.Results:(1)Based on 63 MB rRNA-depletion strand-specific RNA-seq data,24,648 lncRNAs,which have not been annotated in GENCODE and 2759 high-quality circRNAs,are identified of which 82 circRNAs are newly discovered.(2)Through the functional enrichment analysis of the differentially expressed lncRNAs in MB,not only the signal pathways known to function in the genesis and development of MB are discovered,such as central nervous system neuron differentiation,positive regulation of nervous system development,and homophilic cell adhesion via plasma membrane adhesion molecules,etc.,but also discover some new signaling pathways that may participate in the genesis and development of MB,such as the regulation of trans-synaptic signaling,locomotory behavior,and axonogenesis.(3)From WNT,SHH,Group 3 and Group 4 MB,887,818,513,515 subgroup-specific mRNAs,2566,2308,1306,1474 subgroup-specific lncRNAs,and 178,136,52,62 subgroup-specific circRNAs are identified respectively.Fisher’s exact test results shows that the subgroup-specific lncRNA in each subgroup is not only higher in terms of absolute quantity,but also higher in proportion than mRNA and circRNA.(4)Through the GO function enrichment analysis of subgroup-specific lncRNAs,it is not only verified the signal pathways known to be involved in the genesis and development of MB,such as Wnt signaling pathway,blood vessel morphogenesis,light-sensing related pathways,and small GTPases mediated signal transduction,positive regulation of protein kinases activity,heterophilic or homophilic cell adhesion via plasma membrane adhesion molecules,etc.;some new signaling pathways that may be involved in the development of MB have also been discovered,such as SRP-dependent cotranslational protein targeting to membrane,nuclear-transcribed mRNA catabolic process,nonsense-mediated decay,translation initiation,and positive regulation of receptor internalization.(5)By integrating the expression data of mRNA,lncRNA,and circRNA,we construct a co-expression regulatory network and identify the highly associated modules of each subgroup.The proportion of lncRNA in the most relevant module to Group 4 MB is significantly higher than that of other modules,suggesting lncRNA with in module may participate in pathogenesis of MB by coordinating the module key gene GRM8.(6)lncRNA is expressed with higher subgroup specificity than mRNA,which may be due to the stronger regulation effect of DNA methylation on lncRNA.Conclusion:In this study,24,648 lncRNAs which are not annotated in GENCODE are newly discovered in MB through rRNA-depletion strand-specific sequencing and de novo annotation.The functional enrichment analysis of differentially expressed lncRNAs in MB reveal some new signal pathways which may have important functions in the genesis and development of MB,such as the regulation of trans-synaptic signals and axonogenesis.The functional enrichment analysis of subgroup-specific lncRNAs newly discover that SRP-dependent cotranslational protein targeting to membrane,nuclear-transcribed mRNA catabolic process,nonsense-mediated decay may be involved in the genesis and development of MB.The gene co-expression network constructed by integrating mRNA,lncRNA and circRNA expression data provided new information to the understanding of molecular characteristics different subgroup of MB,such as revealing that GRM8 may play an important role in the genesis and development of Group 4 MB.LncRNA expression disorder related to abnormal DNA methylation is one of the possible mechanisms for the genesis and development of MB. |
