Mortaz Esmaeil, Alipoor Shamila D, Tabarsi Payam, Adcock Ian M, Garssen Johan, Velayati Ali Akbar
Clinical Tuberculosis and Epidemiology Research Center, National Research and Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran; Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Clinical Tuberculosis and Epidemiology Research Center, National Research and Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Int J Mycobacteriol. 2016 Dec;5 Suppl 1:S184-S185. doi: 10.1016/j.ijmyco.2016.09.045. Epub 2016 Nov 11.
OBJECTIVE/BACKGROUND: Tuberculosis (TB) is a major global threat to human health, especially in low-income countries. The diagnosis of TB is challenging because of the limitations of specificity and sensitivity with the current diagnostics. Novel, selective biomarkers for TB would be of great practical value. Exosomes are bioactive vesicles with 30-100nm in diameter, which are secreted from almost all cell types and are found in virtually every human body fluid. Exosomes transport micro-RNAs (miRNAs), which are post-transcriptional regulators of gene expression, around the body and allow miRNAs to modulate biological pathways in target cells. Our aim was to investigate the potential of exosomal miRNAs as biomarkers by examining their release from human monocyte-derived macrophages (MDMs) after infection with Mycobacterium using miRNA sequencing.
Human monocytes were obtained from blood and driven to an MDM phenotype using standard protocols. MDMs were infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) or left uninfected as control. Exosomes were collected 72 h postinfection from the cell culture medium and subjected to RNA isolation. Small RNA libraries were constructed and RNA sequencing performed. The raw reads were filtered to eliminate adaptor and primer sequences, and the sequences in FASTQ format were run against the mature human miRNA sequences available in miRBase using BLAST software using a Linux operating system. Micro-RNAs were identified using E=0.01 or 1.
Infection of MDMs with BCG lead to the release of a number of exosomal miRNAs. These mainly consisted with Let-7 family members, miR-155, miR-146a, miR-145, and miR-21 all of which were predicted to target important immune-related genes and pathways.
This study provides evidence for the release of specific miRNAs from BCG-infected MDMs. These results need to be confirmed and the presence of this panel of miRNAs tested in the blood of patients to determine their selectivity and specificity as a diagnostic in patients with TB.
目的/背景：结核病是全球对人类健康的重大威胁，尤其是在低收入国家。由于当前诊断方法在特异性和敏感性方面存在局限性，结核病的诊断具有挑战性。新型的、具有选择性的结核病生物标志物将具有很大的实用价值。外泌体是直径为30 - 100纳米的生物活性囊泡，几乎由所有细胞类型分泌，几乎存在于人体的每一种体液中。外泌体在体内运输微小核糖核酸（miRNA），miRNA是基因表达的转录后调节因子，可使miRNA调节靶细胞中的生物途径。我们的目的是通过使用miRNA测序检查人单核细胞衍生巨噬细胞（MDM）在感染分枝杆菌后外泌体miRNA的释放情况，来研究外泌体miRNA作为生物标志物的潜力。
从血液中获取人单核细胞，并使用标准方案将其诱导为MDM表型。MDM用卡介苗（BCG）感染或作为对照不进行感染。感染后72小时从细胞培养基中收集外泌体，并进行RNA分离。构建小RNA文库并进行RNA测序。对原始读数进行过滤以消除接头和引物序列，并使用Linux操作系统，通过BLAST软件将FASTQ格式的序列与miRBase中可用的成熟人类miRNA序列进行比对。使用E = 0.01或1来鉴定微小核糖核酸。
BCG感染MDM导致释放多种外泌体miRNA。这些主要包括Let - 7家族成员、miR - 155、miR - 146a、miR - 145和miR - 21，所有这些都被预测靶向重要的免疫相关基因和途径。
本研究为BCG感染的MDM释放特定miRNA提供了证据。这些结果需要得到证实，并在患者血液中检测这组miRNA的存在情况，以确定它们作为结核病患者诊断标志物的选择性和特异性。
