Long Noncoding RNAs And Micrornas Regarding Fetal Hemoglobin

Objectives:Long noncoding RNAs（lnc RNAs）and microRNAs（miRNAs）play important roles in gene regulation in a wide aspect of biologic processes and dysregulated expressions of them have been demonstrated being implicated in a variety of human diseases.However,profiles of lncRNAs regarding fetal hemoglobin（HbF）have not yet been reported.The implications of lncRNAs on HbF regulation remain poorly understood.In this study,we surveyed lncRNAs and miRNAs expression profiles in subjects with hereditary persistence of fetal hemoglobin（HPFH）andβ-thalassemia minors with high Hb F levels compared with normal controls using microarray analysis and we performed bioinfor-matics analysis to study the microarray results;certain lncRNAs and their correlative coding genes were selected for further investigation by quantitative reverse transcription polymerase chain reaction（qRT-PCR）,in order to explore lncRNAs associated with Hb F levels and biological functions of lncRNAs in HbF ragulation which may provide new ways for the treatment ofβ-thalassemia major.Methods:1.Microarray was applied for fourteen subjects,seven of them were normal samples,the other were subjects with HPFH andβ-thalassemia minors with high HbF levels.MicroBeads were used to isolate reticulocytes from peripheral blood.Total RNAs were extracted from the enriched reticulocytes.Arraystar Human LncRNA Microarray v4.0 was performed to surveyed lncRNAs and mRNAs expression profiles.About 40173 lncRNAs and 20730 protein-coding transcripts were detected.The original datas from microarray scanner were used for statistical analysis and the differential expression profiles of lncRNAs/mRNAs between the high-HbF and control group were obtained.Through chromosomal localization and BLAST sequence alignment,we analyzed large intervening noncoding RNAs（lincRNAs）and their nearby protein-coding genes（distance<300 kb）.2.The 7^th generation of Exiqon miRCURY LNA?mi RNA Microarray was performed to survey miRNAs expression profiles.The original datas from microarray scanner were used for statistical analysis and the differential expression profiles of miRNAs between the high-HbF and control group were obtained.3.To confirm the microarray data,we selected 3 altered lncRNAs and mi RNAs each for verification.The expression of lncRNAs and miRNAs were detected by qRT-PCR and statistical analysis was used to the expressions of RNAs.The results were compared with the microarray assay.4.Gene Ontology（GO）analysis and KEGG pathway analysis were performed on the aberrantly expressed mRNAs.Coding and non-coding gene coexpression（CNC）networks were constructed based on calculating the Pearson correlation coefficient between coding genes and lncRNAs according to their specific expression levels.The competing endogenous（ceRNA）network was constru-cted using softwares of predicting target genes.5.qRT-PCR was applied for 26 subjects,13 of them were normal samples,the other were subjects with HPFH andβ-thalassemia minors with high HbF levels to investigate the expression of certain lncRNAs including NR₀01589 and T054289,and their associated coding genes.Besides,associations between expression levels of lncRNAs and HbF levels were evaluated base on the Spearman correlation coefficient.Results:1.We filtered expression profiles of lncRNAs/mRNAs and identified aberrantly expressed lncRNAs/mRNAs with absolute fold change of altered expression levels more than 2 and P value less than 0.05.In total,862 lncRNAs and 568mRNAs had aberrantly expressed in the high-HbF group compared with the control group.Of these,605 lncRNAs were up-regulated and 257 were down-regulated;324 mRNAs were up-regulated and 244 were down-regulated.56lincRNAs were aberrantly expressed and nearby coding genes of those lincRNAs had aberrantly expressed at the same time.2.We filtered expression profiles of miRNAs and identified aberrantly expressed mi RNAs with absolute fold change of altered expression levels more than 1.5 and P value less than 0.05.In total,63 miRNAs had aberrantly expressed in the high-HbF group compared with the control group,including 34up-regulated and 29 down-regulated miRNAs.3.Three lncRNAs and three miRNAs were selected for verification of the microarray results by qRT-PCR.The qRT-PCR assay showed that NR₀01589,NR₁20526,T315543,miR-486-3p,miR-19b-1-5p and miR-20a-3p were all up-regulated in the high-HbF group compared with their expression in the controls.The results were consistent with the microarray assay.4.GO analysis showed that the up-regulated mRNAs were involved in the biological processes of iron ion homeostasis,cellular migration and cellular motility,whereas the down-regulated mRNAs were most relevant to regulation of apoptotic process and regulation of cell death.KEGG analysis identified several biological pathways,including Hematopoietic cell lineage and Apoptosis.The CNC networks showed interactions between 167 lncRNAs with HBE1,265lncRNAs with CSF2,CSF3,HLA-DOA and TFRC in the hematopoietic cell lineage pathway,and 140 lncRNAs with GATA1,BECN2,BIRC6 and HTRA2associated with regulation of apoptosis and cell death.5.The ceRNA network showed that 28 up-regulated lncRNAs and 6 down-regulated lncRNAs had the same MREs for miR-486-3p.6.The expressions of NR₀01589 and HBE1 were significantly increased in the high-HbF group compared with the control group and significantly positive correlation was observed between expressions of them.The expressions of T054289 and CSF2 were significantly increased in the high-HbF group and significantly positive correlation was observed between expressions of them.Besides,expression levels of NR₀01589 and T054289 were found to have positive and significant correlations with HbF levels.Conclutions:1.The expressions of several specific lncRNAs and miRNAs were significant differences in subjects with HPFH andβ-thalassemia minors with high HbF levels compared with normal controls.2.Hematopoietic cell lineage and Apoptosis were most important pathways which associated with expression of HbF.3.Six lncRNAs,such as LOC101929207 and G086896,could bind competitive-ly with miR-486-3p,resulting in increased HbF levels.4.NR₀01589 and T054289 were significant correlated with HbF levels.Further studies should be focused on more in-depth investigation of functions of these lncRNAs involved in the HbF regulation.Advance in the knowledge of the Hb F regulation related lncRNAs may be beneficial for the treatment forβ-thalassemia major in the future.