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

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

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7.5×10^(4)(6.0×10^(4),8.8×10^(4)),明显少于NC-mimic组的13.5×10^(4)(12.0×10^(4),14.0×10^(4)),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。

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 7.5×104(6.0×10^(4),8.8×10^(4)),which was significantly lower than that in NC-mimics group(13.5×10^(4)(12.0×10^(4),14.0×10^(4)),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-αincreased 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-αin 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.