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中国防痨杂志 ›› 2021, Vol. 43 ›› Issue (5): 475-481.doi: 10.3969/j.issn.1000-6621.2021.05.012

• 论著 • 上一篇    下一篇

miR-21-3p调节结核分枝杆菌在宿主巨噬细胞内存活机制的研究

吴托雅*, 石金, 郭继东, 刘原园, 逄宇, 鲁洁(), 高飞()   

  1. 010059 呼和浩特,内蒙古医科大学研究生学院 (吴托雅);首都医科大学附属北京胸科医院结核二科(石金),细菌免疫实验室[郭继东(在读研究生)、逄宇];国家儿童医学中心 首都医科大学附属北京儿童医院 北京市儿科研究所耳鼻喉研究室[刘原园(在读研究生)、鲁洁];内蒙古自治区第四医院(高飞)
  • 收稿日期:2020-12-31 出版日期:2021-05-10 发布日期:2021-04-30
  • 通信作者: 鲁洁,高飞 E-mail:lujiebch@163.com;gaofeiwho@163.com
  • 基金资助:
    2019年度首都医科大学科研培育基金(PYZ19043)

Mechanism of miR-21-3p modulating the survival of Mycobacterium tuberculosis in host macrophage

WU Tuo-ya*, SHI Jin, GUO Ji-dong, LIU Yuan-yuan, PANG Yu, LU Jie(), GAO Fei()   

  1. *Graduate School of Inner Mongolia Medical University, Hohhot 010059, China
  • Received:2020-12-31 Online:2021-05-10 Published:2021-04-30
  • Contact: LU Jie,GAO Fei E-mail:lujiebch@163.com;gaofeiwho@163.com

摘要:

目的 研究miR-21-3p在结核分枝杆菌(MTB)感染巨噬细胞免疫应答中的调控作用,并进一步探讨其发挥作用的机制。方法 收集6例临床确诊肺结核患者及6名健康人的外周血,分离出外周血单个核细胞(peripheral blood mononuclear cell,PBMC)。将miR-21-3p模拟物(mimic)、miR-21-3p抑制剂(inhibitor)及阴性对照(NC-mimic及NC-inhibitor)转染细胞后,在不同时间点收集细胞。用MTB标准株H37Rv分别感染细胞系THP-1和U937。采用实时荧光定量逆转录PCR(qRT-PCR)检测 miR-21-3p及促炎因子的表达水平。通过生物信息学工具筛选与miR-21-3p相互作用的靶基因,并采用qRT-PCR验证调控关系。结果 临床标本的检测结果表明,结核病组PBMC的miR-21-3p相对表达量为7.286(6.964,10.483),明显高于健康对照组的1.030(0.997,1.169),差异有统计学意义(U<0.001,P=0.002)。细胞实验结果显示,在感染MTB 24h后,细胞系THP-1及U937中的miR-21-3p相对表达量分别为16.311(15.543,17.030)和72.850(65.343,97.343),与感染前[1.038(0.959,1.165)与1.029(0.979,1.200)]相比,均明显升高,差异均有统计学意义(U值均<0.001,P值均为0.002)。以MTB标准株H37Rv感染miRNA转染的实验组细胞后的菌落形成单位(colony-forming unit,CFU)结果显示,在感染后24h miR-21-3p模拟物组的胞内菌量为7.5×104(6.0×104,8.8×104),明显少于NC-mimic组的13.5×104(12.0×104,14.0×104),差异有统计学意义(U<0.001,P=0.002)。与NC-mimic组相比,miR-21-3p模拟物组巨噬细胞对于MTB感染导致的炎症应答明显增强,IL-6及TNF-α的mRNA相对表达水平分别由1.803(1.729,1.892)及0.960(0.858,1.020)升高至4.520(4.234,5.205)及1.455(1.372,1.523),差异均有统计学意义(U值均<0.001,P值均为0.002);而抑制剂组的白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)相对表达水平分别为0.927(0.901,1.050)及0.781(0.705,0.805),较NC-inhibitor组的1.819(1.007,1.953)及1.101(0.994,1.202)明显降低,差异均有统计学意义(U=2.000,P=0.009;U<0.001,P=0.002)。通过生物信息学工具预测与miR-21-3p序列匹配的基因,并经文献检索,最终筛选出与细胞增殖、凋亡及免疫过程相关的6个候选基因。巨噬细胞分组转染miR-21-3p后,以MTB标准株H37Rv进行感染并检测基因表达情况,结果显示,miR-21-3p模拟物组周期蛋白依赖性激酶8(cyclin-dependent kinases 8,CDK-8)的mRNA相对表达量为0.445(0.434,0.467),明显低于NC-mimic组的1.025(0.917,1.116),差异有统计学意义(U<0.001,P=0.002);抑制剂组CDK-8的mRNA表达水平为1.255(1.185,1.466),明显高于NC-inhibitor组[0.966(0.947,1.042)],差异有统计学意义(U<0.001,P=0.002)。结论 miR-21-3p可抑制MTB在宿主细胞内的生长,在抗结核免疫过程中发挥重要作用。

关键词: 分枝杆菌,结核, 微小RNA, 巨噬细胞, 免疫调节

Abstract:

Objective To investigate the regulatory role of miR-21-3p in the immune response of macrophages infected by Mycobacterium tuberculosis (MTB), and to explore the mechanism. Methods Peripheral blood mononuclear cells (PBMC) were collected from 6 patients with clinically confirmed tuberculosis and 6 healthy subjects. After transfection with miR-21-3p mimic (mimic), miR-21-3p inhibitor (inhibitor) and negative control (NC-mimic and NC-inhibitor), the cells were collected at different time points. The cell lines THP-1 and U937 were both infected by MTB standard strain H37Rv. Real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of miR-21-3p and pro-inflammatory factors. The target genes interacting with miR-21-3p were screened by bioinformatics tools, and the regulatory relationship was verified by qRT-PCR. Results The detection results of clinical specimens showed that the relative expression level of miR-21-3p in PBMC of the tuberculosis group was 7.286 (6.964, 10.483) significantly higher than that of the healthy control group (1.030 (0.997, 1.169), U<0.001, P=0.002). Cell experiment results showed that 24 h after MTB infection, the relative expression levels of miR-21-3p in cell lines THP-1 and U937 were 16.311 (15.543, 17.030) and 72.850 (65.343, 97.343), respectively, which were significantly different from those before MTB infection (1.038 (0.959, 1.165) and 1.029 (0.979, 1.200), both U<0.001, both P=0.002). Colon-forming unit (CFU) results of miRNA transfected cells infected by MTB standard strain H37Rv showed that the intracellular bacteria amount of miR-21-3p mimics group 24 h after infection was 7.5×10 4 (6.0×104, 8.8×104), which was significantly lower than that in NC-mimics group (13.5×104 (12.0×104, 14.0×104), U<0.001, P=0.002). Compared with NC-mimic group, the inflammatory response of macrophages to MTB infection was significantly enhanced in miR-21-3p mimic group, and the mRNA relative expression levels of IL-6 and TNF-α increased from 1.803 (1.729, 1.892) to 4.520 (4.234, 5.205) and 0.960 (0.858, 1.020) to 1.455 (1.372, 1.523), respectively, with statistically significant differences (both U<0.001, both P=0.002). The relative expression levels of IL-6 and TNF-α in inhibitor group were 0.927 (0.901, 1.050) and 0.781 (0.705, 0.805), respectively, which were significantly lower than those in NC-inhibitor group (1.819 (1.007, 1.953) and 1.101 (0.994, 1.202); U=2.000, P=0.009 and U<0.001, P=0.002, respectively). Bioinformatics tools were used to predict the matched genes with miR-21-3p sequences, and literature search was performed to screen out 6 candidate genes related to cell proliferation, apoptosis and immune processes. After macrophage group being transfected bymiR-21-3p, MTB standard strain H37Rv were used for infection and the detection of gene expression was detected. The results showed that the relative mRNA expression of miR-21-3p simulation group cyclin-dependent kinase 8 (CDK-8) was 0.445 (0.434, 0.467), significantly lower than that in the NC-mimic group (1.025 (0.917, 1.116), U<0.001, P=0.002); CDK-8 mRNA expression level in the inhibitor group was 1.255 (1.185, 1.466), significantly higher than that in the NC-inhibitor group (0.966 (0.947, 1.042), U<0.001, P=0.002). Conclusion miR-21-3p could inhibits the growth of MTB in host cells and plays an important role in the anti-tuberculosis immune process.

Key words: Mycobacterium tuberculosis, microRNA, Macrophages, Immunomodulation