高通量测序技术分析急性髓系白血病血浆外泌体微RNA表达谱差异

发布时间：2018-08-27 10:02

【摘要】：急性髓系白血病(acute myeloid leukemia,AML)是一类造血干细胞的恶性克隆性疾病,目前的诊断方法不利于疾病的早期发现,且诊断结果重复性较差。已有大量研究显示,细胞外microRNA(miRNA或miR)富集在外泌体(exosome)中,且受其表面膜的保护而具有很好的稳定性,是理想的分子标志物。目前,多种实体肿瘤均已检测到肿瘤特异性外泌体miRNA(exosomal miRNA)。然而,在AML患者中未见此外泌体miRNA报道。本研究探讨急性髓系白血病血浆外泌体miRNA表达谱差异及新miRNA序列。采用solexa高通量测序技术对7例AML患者(AML组)及7例健康对照者(对照组)血浆外泌体miRNAs进行测序,利用Mireap预测软件进行新miRNAs分析,通过edger差异分析软件筛选组间差异miRNA,获得211个已知的差异表达miRNAs以及2个新miRNAs,选择4个差异表达的miRNAs:miR-155-5p、miR-335-5p、miR-451a及xxx-m0038-5p(新miRNA),在两组(各23例)的血浆外泌体样本中,进行实时荧光定量PCR(qRT-PCR)验证,验证结果与测序结果一致。对差异表达的外泌体miRNA进行靶基因预测及其GO(Gene Ontology)和信号通路富集分析,发现靶基因聚集的生物学功能多数参与生物进展过程的调控。靶基因主要富集在Fox O、MAPK、Hippo信号通路以及HTLV-I感染等。结果显示,AML患者与健康对照者的血浆外泌体miRNA存在着差异性表达。差异性表达的miRNA特异性很高,对进一步阐明AML白血病发生与发展的分子机制、研发新的无创诊断方法、新的诊断标记物和有效治疗AML的方法具有十分重要和深远的意义。
[Abstract]:Acute myeloid leukemia (acute myeloid leukemia,AML) is a malignant clonal disease of hematopoietic stem cells. A large number of studies have shown that extracellular microRNA (miRNA or miR is an ideal molecular marker which is enriched in exocrine (exosome) and is protected by its surface membrane. At present, tumor specific exocrine miRNA (exosomal miRNA). Has been detected in various solid tumors. However, no additional miRNA was reported in patients with AML. The aim of this study was to investigate the difference of miRNA expression profile and new miRNA sequence in plasma exocrine of acute myeloid leukemia (AML). Plasma exocrine miRNAs was sequenced by solexa high-throughput sequencing technique in 7 AML patients (AML group) and 7 healthy controls (control group). New miRNAs analysis was performed with Mireap predictive software. 211 known differentially expressed miRNAs and 2 new miRNAs, selected 4 differentially expressed miRNAs:miR-155-5p,miR-335-5p,miR-451a and xxx-m0038-5p (new miRNA),) in plasma exocrine samples of two groups (23 cases each) were obtained by edger differential analysis software screening of differential miRNA,. Real-time fluorescence quantitative PCR (qRT-PCR) was performed and the results were in good agreement with the sequencing results. Target gene prediction and GO (Gene Ontology) and signal pathway enrichment analysis of differentially expressed exocrine miRNA showed that most of the biological functions of target gene aggregation were involved in the regulation of biological progression. The target genes are mainly concentrated in Fox OMP-MAPK, Hippo signaling pathway and HTLV-I infection. The results showed that there was a differential expression of miRNA in plasma between patients with AML and healthy controls. The specificity of differentially expressed miRNA is very high, which is of great significance for further elucidating the molecular mechanism of the occurrence and development of AML leukemia, developing new noninvasive diagnostic methods, new diagnostic markers and effective methods for the treatment of AML.
【作者单位】：湖南师范大学医学院人体解剖与组织胚胎学教研室;湖南省长沙市中心医院;中南大学湘雅医院血液科;中南大学湘雅二医院老年病学;
【基金】：湖南省自然科学基金项目(No.2015JJ4034)资助~~
【分类号】：R733.71

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