miR-20a-5p在肺结核外周血单核细胞中的表达及临床价值

Expression and clinical value of miR-20a-5p in peripheral blood monocytes from patients with pulmonary tuberculosis

目的:探讨结核菌感染过程中单核细胞miR-20a-5p的表达变化情况及其与肺结核诊断、治疗的关系。方法:选取健康人群(HD)、结核菌潜伏感染(LTBI)、活动性肺结核(PTB)各5例,采集外周血并分离外周血单个核细胞(PBMC),利用免疫磁珠分选外周血CD14+单核细胞,提取总RNA并采取miRNA芯片检测,将肺结核差异表达最为显著的miRNA分子筛选出来,采用定量PCR技术在大样本人群中进行验证(每组各10例),并对5例活动性肺结核患者进行动态随访,利用定量PCR技术检测抗结核治疗前、治疗3个月后、6个月后miR-20a-5p分子的表达情况。利用结核菌强毒株H37Rv、弱毒株H37Ra以及BCG分别感染分化的人单核细胞进行体外实验验证。结果:miRNA芯片显示PTB人群与LTBI、HD人群表达谱存在显著差异,TB人群中共发现14个下调的miRNA分子,包括miR-20a-5p。定量PCR技术证实miR-20a-5p分子在TB人群中的表达水平显著低于HD和LTBI,当服用抗结核药后,其表达水平逐渐上调。用H37Rv、H37Ra及BCG在MOI=10时感染分化的人单核细胞,不同菌株均显著下调miR-20a-5p表达,而以弱毒株下调最为明显。结论:无论是体内还是体外,结核菌感染均下调单核细胞miR-20a-5p表达,而抗结核治疗则显著逆转这种抑制状态。

Objective: To investigate the expression of miR-20A-5P in mononuclear during TB infection and its relationship with the diagnosis and treatment of pulmonary tuberculosis. Methods: The study subjects were recruited and peripheral blood was collected,involving healthy controls (HD, n =5),latent Tuberculosis infection (LTBI, n =5) and PTB ( n =5).PBMC was separated and CD14 + monocytes was sorted by immunomagnetic beads with MACS,then total RNA was extracted and miRNA expression profiles were detected with microarray Chip.The miRNA molecules with the most obvious differential expression in pulmonary tuberculosis were screened out and confirmed in large sample population by quantitative PCR (10 cases in each group).Five patients with active pulmonary tuberculosis were followed up dynamically.The expression of miR-20a-5p was detected by quantitative PCR before treatment and after treatment 3 months and 6 months.Mycobacterium tuberculosis virulent strains,attenuated strain H37Ra and BCG were infected differentiated human mononuclear respectively as vitro experimental validation. Results: miRNA microarray showed significant difference in the expression profiles between PTB and LTBI and HD populations,and there were 14 down-regulated miRNA molecules including miR-20a-5p.miR-20a-5p expression in tuberculosis patients was significantly lower than HD and latent tuberculosis infection confirmed by Real-time Quantitative PCR test,after taking anti-TB drugs,its expression level gradually increased.Differentiated human monocytics were infected with mycobacteria H37Rv,H37Ra and BCG at a MOI = 10,miR-20a-5p expression was significantly down-regulated in different strains,and down-regulation of attenuated strains was most obvious. Conclusion: The expression of miR-20a-5p in monocytes is always down-regulated when infected by mycobacterium tuberculosis both in vitro and in vivo,whereas anti-TB treatment reverse this inhibitory state significantly.