| Objective To investigate the regulatory role of miR-21-3p in the immune response of macrophages infected with Mycobacterium tuberculosis(Mtb),and to further explore the mechanism of its action.Methods Peripheral blood mononuclear cells(PBMC)were collected from patients with pulmonary tuberculosis and healthy subjects.The cells were transfected with miR-21-3p mimic,miR-21-3p inhibitor and negative control,and the cells were collected at different time points.The cell lines THP-1 and U937 were infected with the standard Mycobacterium tuberculosis strain H37 RV,respectively.The expression levels of miR-21-3p and pro-inflammatory factors were detected by quantitative polymerase chain reaction.The target genes interacting with miR-21-3p were screened by bioassay tools,and the negative regulatory relationship was verified by quantitative polymerase chain reaction.Results Retrieving data sets GSE29190 and GSE34608 showed that the expression levels of miR-21-3p in PBMC of TB patients were 5.126(4.387,5.743)and 2965.715(1249.322,1552.253),respectively,which were significantly higher than those of healthy controls(1.043(1.019,1.972)and 701.316(657.595,715.920),with statistical significance(U =2,P=0.0087;U value < 0.001,P value =0.0022);Results of clinical samples showed that the expression level of miR-21-3p in PBMC of TB patients was 7.286(6.964,10.483)significantly higher than that of healthy control group(1.030(0.997,1.169)),and the difference was statistically significant(U < 0.001,P =0.0022).Cell experiment results showed that 24 h after infection with MTB,the expression levels of miR-21-3p in cell lines THP-1and U937 were 16.311(15.543,17.030)and 72.850(65.343,97.343),respectively,compared with 1.038(0.959,1.165)and 1.029(0.979,1.200)at 0 h,and the difference was statistically significant(U value < 0.001,P =0.0022).CFU results of miRNA transfected with H37 RV showed that the amount of intracellular bacteria in the MIMIC group was 7.5(6,8.8)×10424 hours after infection,which was significantly lower than 13.5(12,14)× 104 in the NC-MIMIC group(U < 0.001,P =0.0022).Compared with NC-MIMIC group,the macrophages in MIMIC group significantly enhanced the inflammatory response to MTB infection.The m RNA expression levels of IL-6 and TNF-α increased from 1.803(1.729,1.892)and 0.96(0.858,1.020)to 4.52(4.234,5.205)and 1.455(1.372,1.523),respectively,with statistically significant differences(U < 0.001,P =0.0022;U value < 0.001,P value=0.0022).The expression levels of IL-6 and TNF-α in the inhibitor group were 0.927(0.901,1.050)and 0.781(0.705,0.805),respectively,which were significantly lower than those in the NC-inhibitor group(1.819(1.007,1.953)and 1.101(0.994,1.202),with statistical significance(U =2,P =0.0087;U value < 0.001,P value =0.0022);The m RNA expression level of cyclin-dependent kinases 8(CDK8)in the MIMIC group was 0.445(0.434,0.467),which was significantly lower than 1.025(0.917,1.116)in the NC-MIMIC group(U < 0.001,P =0.0022).CDK8 m RNA 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),the difference was statistically significant(U < 0.001,P =0.0022).Conclusion This study proves that miR-21-3p can inhibit the growth of Mycobacterium tuberculosis in host cells and plays an important role in the anti-tuberculosis immune process. |
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