Genome-wide Characterization Of LncRNAs In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a clonal malignant proliferative disease,which increases the number of myeloid cells in the bone marrow,inhibits the maturation of normal cells,leads to dysfunction of hematopoietic system,finally affects the normal hematopoietic and immune function.Now,the treatment of the disease focuses on the identification of new therapeutic targets,more than 100 gene rearrangements,mutations,etc.were found in AML,but the abnormal molecular markers have not been found in nearly 50%of AML cases.At present,the role of non-coding RNA in AML has become a hot spot.The non-coding RNA accounts for 98%of the total RNA,although they do not encode protein,but the study found that non-coding RNA plays an important role in regulating the growth and development of organisms,the directional differentiation,subcellular distribution and human diseases.Long noncoding RNAs(lncRNAs)are operationally defined as RNA larger than 200 base pairs that appear to lack coding potential.Recently,IncRNAs have emerged as a novel class of pivotal regulators of gene expression and has received increasing attention in the field of AML.In this paper,We do samples with RNA,which are extracted from bone marrow of AML patients and non-leukemia controls,sample labeling and array hybridization were performed.The raw date was analyzed by bioinformatics methods,as described below:the rationality of the experimental design was verified by Principal components analysis(PCA).Through PCA,we found the AML samples and NC samples were located in different regions of space,and six AML samples were relatively concentrated.Differentially expressed analysis showed that 2,616 lncRNAs were significantly differentially expressed between AML and NC samples,among these,1657 IncRNAs were upregulated and 959 lncRNAs were downregulated in AML as compared with NC samples.We performed pathway enrichment analysis for upregulated and downregulated lncRNAs in AML based on their neighboring coding genes.Based on biological processes,the 1657 upregulated IncRNAs were mainly enriched in protein localization,transport and cell cycle.Also,the 959 downregulated lncRNAs were mainly enriched in cell apoptosis and migration,suggesting that these differentially expressed genes may play regulatory role in above pathways.In order tomake a more in-depth study of these differentially expressed genes,we downloaded Reduced representation bisulfite sequencing(RRBS)data in AML patients and normal bone marrow samples from GEO databases to analyzed the changes of DNA methylation in lncRNA promoters,meantime we downloaded human 81 conserved transcription factor binding sites(TFBSs)from UCSC Genome Browser to differentially expressed lncRNA promoters to analyze the distribution of these TFBSs around lncrRNAs.The results showed that there was significant difference in the level of CpG methylation between upregulated lncRNAs and downregulated lncRNAS,and different transcription factor binding events were happened between upregulated lncRNAs and downregulated lncRNAs.Then we studied the interaction between lncRNAs and epigenetic modification in AML,we collected ChIP-Seq data of H3k4me3 and H3k27me3 in five AML patients from the GEO database,found that the two modifications were significantly difference between upregulated lncRNAs and downregulated lncRNAs,around upregulated IncRNAs,the distribution of H3k4me3 was significantly higher,around downregulated lncRNAs the distribution of H3k27me3 was significantly higher.In order to study the position effect of lncRNA and mRNA,we divided the differentially expressed lncRNAs of AML and NC samples into three categories,we identified 120 antisense lncRNAs,96 enhancer lncRNAs,194 lincRNAs,respectively analyzed three types of lncRNAs,found that the expression of these three types of lncRNAs were significantly correlated with the coding gene.At the same time,we randomly selected 4 lncRNAs from these three types of lncRNAs to verify their expression in AML and NC samples with Q-PCR,the results are consistent with array data.Among these lncRNAs,enhancer IncRNA LOC285758 showed the significant association with the poor overall survival of 197 AML samples in the TCGA database,so we choose loc285758 as follow-up function verification.Based on the array data,we assume that loc285758 and HDAC2 can interact with each other,using siRNA knock down technology,this hypothesis is confirmed in AML cells.In conclusion,We provided a novel repository of potential functional lncRNAs for AML research,and discover the new expression pattern of lncRNAs was associated with DNA methylation and transcription factor binding in AML.We revealed the potential regulation of lncRNAs with histone modifications in AML.We revealed three potential interactions between lncRNAs and coding genes in AML and verifide the function and potential regulation betwee lncRNA and HDAC2 in AML cell lines.This has important scientific and clinical value for the future study of the development of AML mechanism and application.