Abstract:

Objective: To investigate the effect and molecular mechanism of miR-17 on affecting the differentiation of initial CD4⁺T cells into regulatory T cell (Treg) and T help cell 17 (Th17) by regulating peripheral blood dendritic cells (DC) of patients with pulmonary tuberculosis. Methods: Peripheral blood samples of 20 patients with pulmonary tuberculosis (tuberculosis group) and 20 outpatient visiting for health check-ups (healthy control group) from Hangzhou Chest Hospital Affiliated to Zhejiang University School of Medicine were collected between February 1 and April 30, 2022.Expressions of miR-17 in DC were detected with qPCR. The mature DC from tuberculosis group were co-cultured with the initial CD4⁺T lymphocytes from healthy control group, then the miR-negative control reagents (miR-negative control group), miR-17 mimic reagents (miR-17 mimic group) and miR-17 inhibitor were added to the cultures, respectively. The differentiation ratios of Treg cells and Th17 cells from the three groups were detected by flow cytometry, and the Eos gene transcription statuses in initial CD4⁺T cells were detected by qPCR and the expressions of Eos were detected by Western blot. Results: The expression of miR-17 in DC of pulmonary tuberculosis patients (12.546±1.572) was significantly higher than that of DC from healthy controls (2.409±1.097,t=28.356,P<0.05). The average proportion of Treg cells in the miR-17 mimic group (4.740%±0.901%) was significantly lower than that in the miR-negative control group (59.235%±4.652%,t=50.755,P<0.01). The average proportion of Th17 cells in the miR-17 mimic group (67.610%±3.495%) was significantly higher than that in the miR-negative control group (27.645%±2.075%,t=38.521, P<0.01). The average proportion of Treg cells in the miR-17 inhibitor group (83.080%±5.770%) was significantly higher than that in the miR-negative control group (59.235%±4.652%, t=14.988, P<0.01); the average proportion of Th17 cells in the miR-17 inhibitor group (11.405%±1.777%) was significantly lower than that in the miR-negative control group (27.645%±2.075%, t=27.044, P<0.01).The transcription level of Eos mRNA gene in the initial CD4⁺T lymphocytes in the miR-17 mimic group (0.181±0.123) was down-regulated to 38.4%, which was significantly lower than that in the miR-negative control group (0.471±0.217, t=10.449, P<0.05). The transcription level of Eos mRNA gene in the initial CD4⁺T lymphocytes in the miR-17 inhibitor group (0.889±0.295) was up-regulated to 1.887-fold, significantly higher than that in the miR-negative control group (0.471±0.217,t=16.635, P<0.05).The relative expression of Eos in the initial CD4⁺T lymphocytes of the miR-17 mimic group (3.626±1.319) was reduced to 77.7%, significantly lower than that in the miR-negative control group (4.664±1.456,t=8.528, P<0.05). The relative expression of Eos in the initial CD4⁺T lymphocytes in the miR-17 inhibitor group (6.148±1.701) increased to 1.318-fold, significantly higher than that in the miR-negative control group (4.664±1.456,t=8.035, P<0.05). Conclusion: DC miR-17 regulates the initial CD4⁺T lymphocytes unevenly differentiating to Treg/Th17, thus participates in the process of Mycobacterium tuberculosis infection.

Key words: Mycobacterium tuberculosis, Dendritic cells, T-lymphocytes, MicroRNA