ESAT-6 Modulates Macrophage Apoptosis in Mycobacterium Tuberculosis via lncNEAT1/miR-125b-5p/TNF-α Pathway

Zulipikaer Abudureheman,1,* Hui Gong,1,* Tuerhongjiang Axirejiang,2,* Jingran Xu,1 Abudushalamu Abuduwake,2 Ayiguli Alimu,1 Meiheriban Mamuti,1 Aifang Zheng,3 Li Li3 1Department of Clinical Research Center of Infectious Diseases (Tuberculosis), First People’s Hospital of Kashi, Kashi, XinJiang, People’s Republic of China; 2Department of Dermatology, First People’s Hospital of Kashi, Kashi, XinJiang, People’s Republic of China; 3Department of Respiratory and Critical Care Medicine, First People’s Hospital of Kashi, Kashi, XinJiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li Li, Email [email protected]: Tuberculosis (TB), resulting from the bacterial pathogen Mycobacterium tuberculosis (Mtb), continues to be a leading cause of death and illness globally. Mtb employs secretory proteins to avoid host immune responses during the infection process and is able to survive, spread and replicate within the hostile micro-environment. Early secreted antigenic target 6 kDa (ESAT-6), the major virulence factor of Mtb, plays an important role in Mtb-induced macrophage apoptosis, which could benefit the dissemination of Mtb. However, the underlying mechanism of ESAT-6 in macrophage apoptosis still unclear.Methods: In this research, the human monocytic leukemia cell lines (THP-1) were treated with Phorbol 12-myristate 13-acetate (PMA) to differentiation into M0 macrophages, and the role of recombinant ESAT-6 in macrophage apoptosis was investigated using Annexin V-FITC/propidium iodide (PI) assay with Flow CytoMetry analysis. The small interfering RNA (siRNA) of Long non-coding RNA nuclear enriched abundant transcript 1 (lncNEAT1) was used to silencing the RNA expression of lncNEAT1. The expression level of lncNEAT1, microRNA-125b-5p (miR-125b-5p) and tumor necrosis factor-alpha (TNF-α) mRNA were investigated using RT-qPCR technique. Additionally, Western blot was used to detect the protein expression of TNF-α. Furthermore, the downstream regulating mechanisms of lncNEAT1 were investigated using bioinformatics analyses and luciferase reporter assays.Results: The results showed that ESAT-6 induces macrophage apoptosis in a dose-dependent manner via upregulation of the lncNEAT1 and lncNEAT1 can target miR-125b-5p, while miR-125b-5p can also target the 3′UTR (Untranslated Regions) of TNF-α mRNA. Moreover, inhibition of lncNEAT1 alleviated ESAT-6 induced macrophage apoptosis by targeting miR-125b-5p/TNF-α axis.Discussion: The results of this study indicated that ESAT-6 induces macrophage apoptosis by regulating lncNEAT1/miR-125b-5p/TNF-α pathway, which may provide a possible therapeutic target for the treatment of TB.Keywords: apoptosis, ESAT-6, macrophage, Mycobacterium tuberculosis, tuberculosis