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

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FoxO、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。

Analysis of Differential Expression Profiles of Exosomal MicroRNA in Acute Myeloid Leukemia through High-Throughput Sequencing Technologies

Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells. The current diagnostic method is not conducive to the early detection of the disease. The diagnostic results are poorly reproducible. Many studies have shown that extracellular miRNAs (miRNAs) are enriched in exosomes and are well protected by membrane protection. Tumor-specific exosomal miRNAs have been found in many solid tumors. However, it is not reported in AML patients. We showed the differences of miRNAs expression profiles and new miRNA sequences of exosomes in AML were studied. High throughput sequencing technology was used to detect the expression of exosomal miRNAs in 7 AML patients and 7 matched healthy controls. Using miRNA database tools, Mireap and Edger software analysis, 211 differentially expressed miRNAs and 2 new miRNAs were identified (P<0.05). Four miRNAs (miR-155-5p, miR-335-5p, miR-451a and a new miRNA) were identified and analyzed by qRT-PCR analysis. In addition, target genes of differentially expressed exosomal miRNAs were predicted. GO (Gene Ontology) and signal pathway enrichment analysis of target genes were conducted with DAVID. The biological function of target gene aggregation was involved in the regulation of biological process. Target genes were mainly enriched in FoxO, MAPK and Hippo signaling pathway and HTLV-1 infection. The results showed that there were differentially expressed miRNAs in plasma exosomes of AML patients and healthy controls, and the specificity of differentially expressed miRNAs was high. Our study provided understanding of the molecular mechanism of AML development and progression, which may help to find new non-invasive diagnostic methods, new diagnostic markers and effective treatment of AML.