Identification Of Non-coding Diagnostic Elements And Reveals The Epigenetic Regulatory Roles In Brain Complex Diseases

Objective: DNA methylation is one of the fundamental features of epigenetic regulation and has been reported to play important roles in the brain complex diseases.Meanwhile,alterations of DNA methylation can affect the activities of various non-coding element,which included long non-coding RNAs(lncRNAs)and enhancer.However,a systematic analysis of the regulatory mechanisms of non-coding elements which mediated by DNA methylation in brain diseases is still lacking.This study focused on dissecting the immune regulatory function of lncRNAs in glioma and constructing an enhancer regulatory network in Alzheimer’s disease,to systematically explore the abnormality of DNA methylation in non-coding regions.The completion of this study will provide new insights into the pathogenesis and the identification of novel diagnostic markers for brain diseases.Methods: We first collected 551 adult glioma samples with paired epigenetic and transcriptomic data from The Cancer Genome Atlas(TCGA)database,covering both low-grade gliomas and glioblastoma multiforme patients.The glioma samples were clustered into different molecular subtypes based on the lncRNA methylation features.Immune subtypes were defined based on the activities of immune pathways,immune signatures,antitumor immunoactivity,and immune cell infiltration.The differences for clinical molecular and epigenetic features were also explored.Next,epigenetically regulated(ER)lncRNAs were identified by integrating DNA methylation and lncRNA expression,potential molecular functions were further explored through e Forge and Metascape tools.To clarify the immune regulatory function of ER lncRNAs,computational methods such as TIMER and Imm Lnc were used to evaluate the association of lncRNAs with immune cell infiltration and immune pathways.Finally,the role of lncRNA on the development and progression of glioma was determined by in vitro experiments including CCK8,si RNA,cell scratching,and transwell assay.Next,we comprehensively constructed and systematically analyzed the enhancer regulatory network which mediated by DNA methylation in AD disease.First,DNA methylation 450 K microarray in different brain regions of AD patients were obtained from the Gene Expression Omnibus(GEO),including 1997 samples.Combining DNA methylation profiles with enhancer annotation,we identified enhancers that are epigenetically regulated in AD and dissect their functions based on nearest protein-coding genes.Enhancer regulatory networks in different brain regions of AD were constructed by integrating enhancer sequences,Hi-C interactions and methylation levels.Methy CIBERSORT was used to calculate the cellular composition of the brain microenvironment,and further explored the its relationship with enhancer regulatory networks.Furthermore,through integrating sn ATAC-seq and sn RNA-seq data and enhancer regulatory networks from AD frontal lobe,we characterized the chromatin accessibility status of enhancers,and finally prioritied enhancer regulatory triples in AD specific cell types.RESULTS: We identified 2 robust glioma subtypes exhibited distinct immune-related characteristics.The“hot” glioma subtype(C1 cluster)has global demethylation properties,which is regulated by the APOBEC3 gene family.A total of149 ER lncRNAs were identified in glioma immune subtypes and involved in brain development and immune system regulation.Among them,29 ER lncRNAs were not only associated with 6 types of immune cell infiltration but also involved in the regulation of immune pathway activity.In addition,patients in the C1 subtype have poor prognosis and featured by immune escape characteristics.By integrating the glioma anti-PD1 immunotherapy dataset,AC131097.3,CD109-AS1,LINC02447 and LINC01765 were found to exhibit the potency to be novel glioma immune evasion markers.Through in vitro experiments,we confirmed that CD109-AS1 and LINC02447 can promote the proliferation,migration and invasion of glioma cells,and the protein activities of immune escape markers FOXP3,PD-L1 and CTLA4 were significantly decreased after lncRNA interference.These results revealed the oncogenic role of epigenetically activated lncRNAs.Next,through the analysis of epigenetic datasets for AD patients,we found that DNA methylation alterations(DMA)of enhancers were widespread in different brain regions of AD,and DMA enhancers were associated with brain development and cognitive function.By constructing enhancer regulatory network,we found essential transcription factors like ATF3 and FOSL1 interact with enhancers in multiple brain regions.The composition of the brain microenvironment,including glial cells,neurons,and monocytes et.al,was altered in AD tissues.Moreover,enhancer regulatory networks were significantly associated with brain microenvironment.Sn ATAC-seq and sn RNA-seq analysis observed the altertions of chromatin accessibility activity at brain-specific enhancers as well as DMA enhancers regions.Finally,hyper-methylated triple enh＿4341-SOX15/SOX21-ANKRD44 and the hypo-methylated triple enh＿199-BACH2-KAZN were prioritied in AD microglia and oligodendrocytes.The methylation of enhancer is closely related to the pathological state of AD.Conclusion: Taken together,our study systematically characterized the epigenetic non-coding element landscape in brain complex diseases,and reveals the crosstalks among DNA methylation,non-coding elements,downstream target genes and immune function.Based on bioinformatics analysis,molecular biology experiments demonstrated that the ER lncRNA CD109-AS1 and LINC02447 served as novel immune escape markers for glioma.The findings of our work will provide novel diagnostic markers and epigenetic therapeutic targets for brain diseases such as glioma and AD.