上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2020, Vol. 40 ›› Issue (2): 171-.doi: 10.3969/j.issn.1674-8115.2020.02.005

• 论著·基础研究 • 上一篇    下一篇

血浆外泌体来源的miRNA-323a-3p作为结核病潜在生物标志物的评估

林 砺1，李海波2，夏 凡3，周计雪3，郭晓奎1，张舒林1, 2   

  1. 1. 上海交通大学基础医学院免疫学与微生物学系，上海 200025；2.上海市（复旦大学附属）公共卫生临床中心，上海 201508；3.中国人民解放军第905医院肺科，上海 200235
  • 出版日期:2020-02-28 发布日期:2020-03-20
  • 通讯作者: 张舒林，电子信箱：biolotus@163.com。郭晓奎，电子信箱：xkguo@shsmu.edu.cn。为共同通信作者。
  • 作者简介:林 砺（1994—），女，硕士生；电子信箱：18222952882@163.com。
  • 基金资助:
    2019 年度“技术转移推广项目” (ZT201917）；国家自然科学基金（81871613）。

Evaluation of exosome-derived miRNA-323a-3p human plasma as a potential biomarker for tuberculosis

LIN Li, LI Hai-bo, XIA Fan, ZHOU Ji-xue, GUO Xiao-kui, ZHANG Shu-lin   

  1. 1. Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China; 2. Shanghai Public Health Clinical Center, Shanghai 201508, China; 3. Department of Pulmonology, the 905th Hospital of the Chinese Peoples Liberation Army, Shanghai 200235, China
  • Online:2020-02-28 Published:2020-03-20
  • Supported by:
    Technology Transfer Promotion Project in 2019 (ZT201917); National Natural Science Foundation of China (81871613).

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摘要： 目的·研究血浆外泌体来源的microRNA（miRNA），为结核病的诊断寻找潜在标志物。方法·采用卡介苗（Bacille Calmette-Guérin，BCG，即减毒牛分枝结核杆菌）感染人单核细胞白血病细胞（THP-1）来源的巨噬细胞（BCG组），设置未感染细胞为对照组。利用超速离心法获取2组细胞培养上清液中的外泌体，通过透射电子显微镜（transmission electron microscope，TEM）、蛋白质印迹法进行形态学及蛋白标志物鉴定，并对外泌体进行转录组测序分析。选取测序结果中的3个差异表达miRNAs，就10例结核病患者（结核病组）和8例健康志愿者（健康对照组）的血浆外泌体进行临床验证。通过TargetScan、miRDB和PicTar数据库对存在潜在差异表达的miRNA进行靶基因预测。结果·细胞培养上清液外泌体测序结果显示，与对照组相比，miRNA-323a-3p、miRNA-29a-3p和miRNA-29b-3p在BCG组的表达均有显著差异，而在临床验证中仅有miRNA-323a-3p在2组表达间的差异具有统计学意义（P0.004）；且miRNA-323a-3p在结核患者血浆来源的外泌体中显著下调，与转录组结果一致。结论·血浆外泌体来源的miRNA-323a-3p在结核患者中的表达显著下调，且其靶基因与自噬作用密切相关，有望为结核病的机制研究提供新思路。

关键词: 外泌体, 微小RNA, miRNA-323a-3p, 结核, 超速离心法

Abstract:

Objective · To study the microRNA (miRNA) derived plasma exosomes, and to find the potential biomarker for the diagnosis of tuberculosis. Methods · Macrophages [Bacille Calmette-Guérin (BCG) group] human monocytic leukemia cells (THP-1) were infected with BCG, and uninfected macrophages were set as control group. Exosomes the cell culture supernatant of the two groups were obtainedultracentrifugation. The morphology and protein markers of exosomes were identifiedtransmission electron microscopy (TEM) and Western blotting. Total RNA of exosomes was analyzedtranscriptome sequencing. Three miRNAs with different were selected the sequencing results, and the plasma exosomes of 10 patients with tuberculosis (tuberculosis group) and 8 healthy volunteers (healthy control group) were clinically verified. The target genes of miRNAs with potential differential were predictedTargetScan, miRDB and PicTar databases. Results · The sequencing results of exosomes the cell culture supernatant showed that compared with the control group, the of miRNA-323a-3p, miRNA-29a-3p and miRNA-29b-3p in BCG group were significantly different. In clinical verification, only miRNA-323a-3p between the tuberculosis group and the healthy control group was statistically significant (P0.004). miRNA-323a-3p was significantly down-regulated in the plasma-derived exosomes of tuberculosis patients, which was consistent with the results of sequencing results. Conclusion · The of miRNA-323a-3p plasma exosomes in patients with tuberculosis is significantly down-regulated, and its target gene is closely related to autophagy, which is expected to provide new ideas for the mechanism of tuberculosis.

Key words: exosome, micro RNA (miRNA), miRNA-323a-3p, tuberculosis, ultracentrifugation