Objective To analyze Mycobacterium tuberculosis ( M. tb) strain H37Rv induced differential expression miRNAs from host macrophage, and we explore the diagnosis value of M.tb infection as a biomarker in clinic.
Methods The Mycobacter tuberculosis (M.tb) strain H37Rv was incubated and cultured with human macrophage line THP-1. The total RNA was extracted from the THP-1 cells in 12 h, 24 h and 48 h. The miRNA of groups was detected by miRNA microarray chips and the differential expressed miRNAs were analyzed for verification experiments furtherly. The rt-PCR was used to detect the differential expressed miRNAs from infected THP-1 by M.tb strains, isolated from 120 clinical sputum samples, respectively. Following, the RNA was extracted from sputum sample to detect the miRNA using rt-PCR and this detecting efficiency of several methods was compared.
Results The data showed the expression level of six miRNA have a significant change, that miR-155, miR-29b-1*, miR-150, miR-146a, miR-212 and miR-483-5p. The six miRNAs were detected from THP-1 cells, were infected by 120 clinical M. tb strains from different areas for 24 h. The results showed miR-155 was a higher level than others in the tests. After that the miR-155 as a biomarker for M.tb infection diagnosis, the RNA was extracted from 68 sputum samples to detect the level of miR-155. The sensitivity and specificity of miR-155 detectionwere 94.1% and 93.8%, respectively. The sensitivity was higher than that of sputum smear in 22.1% and tuberculosis antibody in 83.8% significantly ( P<0.05). But there was no significant difference with ELISPOT in 97.1% ( P>0.05). The ranking of sputum smear was positive correlation with miR-155 detection in sputum ( R2=0.743, P<0.05).
Conclusions The data suggested that miR-155 detection is a viral significance for active TB infection diagnosis and bacteria-loads evaluation indirectly as an efficiency biomarker.
摘要： 目的 分析结核分枝杆菌 H37Rv 感染诱导宿主巨噬细胞表达 miRNA 的变化, 进而验证差异 miRNA 对结核 分枝杆菌感染的临床诊断意义。 方法 使用 H37Rv 菌株感染人巨噬细胞系 THP-1 细胞后, 收集 12 h、24 h 和 48 h 细胞 并提取总 RNA, 进行 miRNA 微阵列芯片分析; 再将临床分离的 120 株不同个体来源的结核分枝杆菌菌株与 THP-1 细胞 共培养, 使用实时定量 PCR (real time-PCR, rt-PCR)验证差异 miRNA 的表达水平; 随后收集结核患者的痰液标本处理 后, 提取总 RNA, 进行实时荧光定量 PCR 检测痰液中的差异表达的 miRNA 水平。 结果 H37Rv 体外刺激 THP-1 细胞, 使用 miRNA 微阵列芯片分析, 结果显示：miR-155、miR-29b-1*、miR-150、miR-146a、miR-212 和 miR-483-5p 共计 6 个 miRNAs 水平在 3 个时间点升高均较为明显; 将来自不同区域的 120 株结核菌株分别与 THP-1 细胞的共培养 24 h, 检测 发现 miR-155 表达水平最高; 以 miRNA-155 为检测靶标, 对 68 例结核患者的痰液进行检测, 灵敏度和特异度分别为 94.1% 和 93.8%。其灵敏度高于痰涂片的 22.1% 和结核抗体的 83.8% , 差异具有统计学意义 ( P<0.05) 。但与 ELISpot 法 的 97.1%, 差异无统计学意义 ( P>0.05) 。在对痰涂片阳性结果与 miRNA 检测结果相关性分析中, 发现 miR-155 其表达 水平与痰涂片阳性等级呈正相关 ( R2=0.743, P<0.05) 。 结论 此研究提示 miR-155 能够作为活动性结核诊断的有效靶 标, 且对间接评估患者肺部荷菌量有重要意义。