Mechanistic study of miR-207 regulation of Mycobacterium tuberculosis survival in macrophages
10.3969/j.issn.1671-7856.2024.12.005
- VernacularTitle:miR-207调控结核分枝杆菌在巨噬细胞内存活的机制研究
- Author:
Wenya DU
1
;
Yumei DAI
1
;
Linzhi YUE
1
;
Tao MA
1
;
Lixian WU
1
Author Information
1. 大理大学基础医学院微生物学与免疫学教研室,云南大理 671000
- Publication Type:Journal Article
- Keywords:
Mycobacterium tuberculosis;
H37Ra;
miR-207;
autophagy;
macrophage;
apoptosis;
pyroptosis
- From:
Chinese Journal of Comparative Medicine
2024;34(12):41-49
- CountryChina
- Language:Chinese
-
Abstract:
Objective miR-207 is differentially expressed in many diseases.We investigated the mechanism by which miR-207 overexpression regulates the survival of Mycobacterium tuberculosis(H37Ra)in macrophages,to provide a theoretical basis for the targeted therapy of tuberculosis.Methods Macrophages were divided into four groups:blank(Ana-1 cells),control(cells infected with H37Ra),mi(infected with H37Ra and transfected with miRNA-207 mimics),and mi-NC(infected with H37Ra and transfected with NC mimics)groups.A model of tuberculosis infection was established using H37Ra-infected Ana-1 cells,and miRNA-207 and NC mimics were transfected into Ana-1 cells using the liposome transfection method.Tuberculosis colony-forming units were counted to assess the effect of miR-207 on intracellular mycobacterial load and clearance of extracellular residual mycobacteria.The total apoptosis rate was detected by flow cytometry.The relative expression levels of miR-207 and apoptosis,pyroptosis,inflammation,and autophagy genes were measured by quantitative real-time polymerase chain reaction(qPCR).Relative expression levels of apoptosis,pyroptosis,and autophagy proteins were detected by Western blot.Fluorescence microscopy and multifunctional enzyme labeling were used to detect the fluorescence intensity of intracellular reactive oxygen species(ROS)and lactate dehydrogenase(LDH).Results Successful establishment of the infection model was observed under the microscope.qPCR showed that miR-207 expression was lower in the control compared with the blank group(P＜0.01),indicating differential expression between these two groups.miR-207 expression was significantly higher in the mi compared with the mi-NC group(P＜0.0001),indicating successful establishment of the transfection model.The number of colonies and total apoptosis were both higher in the mi group compared with the mi-NC and control groups(P＜0.001).qPCR and Western blot showed that the relative expression levels of apoptotic genes and proteins were higher in the control group than in the blank group(P＜0.05),and higher in the mi group than in the mi-NC group(P＜0.05).The relative expression levels of inflammatory genes were higher in the control than in the blank group(P＜0.001).The relative expression levels of inflammatory genes were higher in the mi group than in the mi-NC group(P＜0.05),and the relative expression levels of pyroptosis genes and proteins were higher in the control group compared with the blank group(P＜0.01)and higher in the mi group compared with the mi-NC group(P＜0.05).The relative expression levels of the autophagy positively-regulated genes LC3 and Beclin1 were higher in the control compared with the blank group(P＜0.0001),and lower in the mi than in the mi-NC group(P＜0.05),while negatively-regulated autophagy genes showed the opposite trend.Autophagy-related proteins showed similar trends to the autophagy genes.ROS fluorescence intensity was higher in the control compared with the blank group(P＜0.05),and higher in the mi compared with the mi-NC group(P＜0.001).LDH content was higher in the control than in the blank group(P＜0.01),but there was no significant difference between the mi and mi-NC groups(P＞0.05).Conclusions miR-207 overexpression promotes apoptosis,cellular pyroptosis,and inflammation,inhibits autophagy,and favors H37Ra survival.These result provide a potential new direction for the treatment of tuberculosis.
