Systematic Characterization Of LncRNAs’ Cell-to-Cell Expression Heterogeneity In Glioblastoma Cells

Objective: Glioblastoma(GBM)is the most common malignant adult brain tumor generally associated with high level of cellular hetero geneity and a dismal prognosis.GBM has now manifested its heterogeneous nature in many ways.It is,however,becoming increasingly clear that intratumoral genetic heterogeneity is central to GBM biology,potentially posing a great c hallenge to effective treatment.Originally,intratumoral heterogeneity has been verified via the analysis of bulk tumors revealing regional copy number variation(in EGFR,PDGFRA and PTEN),heterogeneous somatic mutations(in TP53)or gene expression difference(in MGMT).Further supportive evidence comes from the observation that spatially distinct fragments sampled from the same tumor corresponded to different GBM molecular subtypes.These findings represent an important step toward understanding intratumoral heterogeneity,but they deserve closer scrutiny at higher resolution because each cell within a single tumor possibly possesses a unique gene expression signature under specific conditions.Long noncoding RNAs(lnc RNAs),which are a type of noncoding RNA(nc RNA)varying in size from 200 bp to >100 kb,are transcribed by RN A polymerase II,and are often spliced and polyadenylated.They have been identified by a variety of methods and a growing number of specific lnc RNAs have been demonstrated to affect genomic functions,including imprinting,enhancer function,X-chromosome inactivation,chromatin structure and genomic rearrangements during the generation of antibody diversity.lnc RNAs can exert their effects through mechanisms such as chromatin remodeling,cis regulation at enhancers and post-transcriptional regulation of mRNA processing.Thus,they have been proposed as key mediators of diverse biological processes including cell pluripotency and tumorigenesis.Currently,accumulated evidence demonstrates that some lnc RNAs,often aberrantly expressed in GBM,have been implicated in histological/molecular subtypes and malignant phenotypes,thereby possessing potentials as biomarkers for diagnosis and prognosis,and as therapeutic targets.Long noncoding RNAs(lnc RNAs)are emerging as novel mediators of tumorigenesis.Recently developed single-cell RNA-seq provides an unprecedented way for analysis of the cell-to-cell variability in lnc RN A expression profiles.The purpose of this paper is to study the cha racterization of lnc RN A expression heterogeneity lays the foundation for future efforts to further understand the function of lnc RNA,develop valuable biomarkers,and enhance knowledge of GBM biology.Methods:(1)RN A expression datasets and the corresponding sample information were downloaded from NCBI GEO Data Sets websites(http://www.ncbi.nlm.nih.gov/gds/)with accession no.GSE57872.(2)The transcriptome used for mapping contains 80,216 high-confidence lnc RNA transcripts corresponding to 48,028 lnc RN A genes from LNCipedia 3.0 and all protein-coding genes of Ensembl(version 74).Bowtie(version 1.1.1)was used to map paired-end 25-bp reads.Sellect the research object form the above mentioned results by quality control and higher credibility.Then analysis the relative expression of them.(3)Principal component analysis was performed with lnc RNAs and observes the transcriptional diversity within each single cell from tumors.For SOM construction,the 500 lnc RNA genes(from 2,003 lnc RNAs)or 2,383 genes(from 7,148 genes)with the greatest variance among the samples were first used for training a SOM to observe the transcriptional diversity within each single cell from tumors.(4)Stemness signature was defined as the set of lnc RNAs whose expression was higher in GSC cultures compared with the corresponding differentiated glioma cell(DGC).(5)The list of subtype identifier lnc RNAs was collected from.Thirty-one lnc RNAs with the greatest variance among all single cells were used for subtype analysis.The results was compared with the classification scheme of GBM subtypes established by The Cancer Genome Atlas(TCGA).Results: Here we comprehensively examined the expression patterns of 2,003 lnc RNAs in 380 cells from five primary GBMs and two gliob lastoma stem-like cell(GSC)lines.Employing the self-organizing maps,we displayed the landscape of the lnc RNA expression dynamics for individual cells.Further analyses revealed heterogeneous nature of lnc RN A in abundance and splicing patterns.Moreover,lnc RNA expression variation is also ubiquitously present in the established GSC lines composed of seemingly identical cells.Through comparative analysis of GSC and corresponding differentiated cell cultures,we defined a stemness signature by a consensus set of 31 differentially expressed lnc RNAs,which can disclose stemness gradients in five tumors.Additionally,based on known lnc RNAs specific to subtypes,each tumor was found to comprise individual cel s representing four subtypes.Conclusions: Our systematic characterization of lnc RNA expression heterogeneity lays the foundation for future efforts to further understand the function of lnc RN A,develop valuable biomarkers,and enhance knowledge of GBM biology.