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.
Wechat
京公网安备11010202007215号
Total visitors: Visitors of today: Now online:
This work is licensed under Creative Commons Attribution 3.0 License.