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<p>Pulmonary tuberculosis (TB), caused by mycobacterium tuberculosis (M.tb) infection, remains a major global health threat. Macrophages serve as the primary host cells for M.tb, and the bacterium’s ability to manipulate macrophage function is critical for its survival and pathogenesis. However, the specific molecular mechanisms regulating M.tb-induced macrophage injury require further elucidation. This study aimed to screen key regulatory genes and investigate the underlying mechanisms involving m6A modification in M.tb-induced THP-1 macrophages. Differentially expressed genes (DEGs) between M.tb-induced THP-1 macrophages and normal THP-1 macrophages were identified through bioinformatics analysis of the GSE203261 dataset. Expression levels of the target gene were validated in clinical blood samples and M.tb-induced THP-1 cells via quantitative real-time PCR (qRT-PCR) and Western blotting. Loss-of-function experiments were performed using small interfering RNA (siRNA), while overexpression plasmids were utilized for gain-of-function studies. The association between N6-methyladenosine (m6A) modification and the target gene was investigated using methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), and dual-luciferase reporter assays. Cell viability, apoptosis, inflammatory cytokine levels, and oxidative stress markers were assessed to evaluate cellular injury, and flow cytometry was employed to analyze M1 macrophage polarization. Colony-forming unit (CFU) assay was performed to assess the survival of M.tb. NF-κB pathway-related markers were detected by Western blotting. Sialic acid binding Ig like lectin 1 (SIGLEC1) was identified as a significantly upregulated gene in M.tb-induced THP-1 macrophages, and its high expression was confirmed in the blood of pulmonary TB patients and M.tb-induced THP-1 macrophages. Knockdown of SIGLEC1 attenuated M.tb-induced injury by enhancing cell viability, suppressing apoptosis, reducing the levels of inflammatory cytokines (IL-6, TNF-α), reactive oxygen species (ROS), and malondialdehyde (MDA), and increasing superoxide dismutase (SOD) levels. Additionally, SIGLEC1 silencing inhibited M1 polarization. Mechanistically, the methyltransferase-like 3 (METTL3) was found to bind to SIGLEC1 mRNA and enhance its stability in an m6A-dependent manner, a process dependent on the reader protein insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2). Rescue experiments demonstrated that while METTL3 knockdown alleviated M.tb-induced macrophage injury and inactivated the NF-κB pathway, these protective effects were reversed by SIGLEC1 overexpression. This study elucidated a novel METTL3/IGF2BP2/SIGLEC1 regulatory axis that mediated M.tb-induced macrophage injury and M1 polarization via the NF-κB signaling pathway. These findings highlight the potential of SIGLEC1 as a promising biomarker and provide a rationale for its further investigation as a therapeutic target for pulmonary TB.</p>

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Screening of SIGLEC1 based on transcriptomics and investigation of its function and mechanism in regulating mycobacterium tuberculosis-induced injury and polarization in THP-1 macrophages

  • 期刊论文

  • 发表日期：

    2026年6月26日

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  • Jianwu Liu,
  • Guolin She,
  • Haijian Wang

摘要

Pulmonary tuberculosis (TB), caused by mycobacterium tuberculosis (M.tb) infection, remains a major global health threat. Macrophages serve as the primary host cells for M.tb, and the bacterium’s ability to manipulate macrophage function is critical for its survival and pathogenesis. However, the specific molecular mechanisms regulating M.tb-induced macrophage injury require further elucidation. This study aimed to screen key regulatory genes and investigate the underlying mechanisms involving m6A modification in M.tb-induced THP-1 macrophages. Differentially expressed genes (DEGs) between M.tb-induced THP-1 macrophages and normal THP-1 macrophages were identified through bioinformatics analysis of the GSE203261 dataset. Expression levels of the target gene were validated in clinical blood samples and M.tb-induced THP-1 cells via quantitative real-time PCR (qRT-PCR) and Western blotting. Loss-of-function experiments were performed using small interfering RNA (siRNA), while overexpression plasmids were utilized for gain-of-function studies. The association between N6-methyladenosine (m6A) modification and the target gene was investigated using methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), and dual-luciferase reporter assays. Cell viability, apoptosis, inflammatory cytokine levels, and oxidative stress markers were assessed to evaluate cellular injury, and flow cytometry was employed to analyze M1 macrophage polarization. Colony-forming unit (CFU) assay was performed to assess the survival of M.tb. NF-κB pathway-related markers were detected by Western blotting. Sialic acid binding Ig like lectin 1 (SIGLEC1) was identified as a significantly upregulated gene in M.tb-induced THP-1 macrophages, and its high expression was confirmed in the blood of pulmonary TB patients and M.tb-induced THP-1 macrophages. Knockdown of SIGLEC1 attenuated M.tb-induced injury by enhancing cell viability, suppressing apoptosis, reducing the levels of inflammatory cytokines (IL-6, TNF-α), reactive oxygen species (ROS), and malondialdehyde (MDA), and increasing superoxide dismutase (SOD) levels. Additionally, SIGLEC1 silencing inhibited M1 polarization. Mechanistically, the methyltransferase-like 3 (METTL3) was found to bind to SIGLEC1 mRNA and enhance its stability in an m6A-dependent manner, a process dependent on the reader protein insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2). Rescue experiments demonstrated that while METTL3 knockdown alleviated M.tb-induced macrophage injury and inactivated the NF-κB pathway, these protective effects were reversed by SIGLEC1 overexpression. This study elucidated a novel METTL3/IGF2BP2/SIGLEC1 regulatory axis that mediated M.tb-induced macrophage injury and M1 polarization via the NF-κB signaling pathway. These findings highlight the potential of SIGLEC1 as a promising biomarker and provide a rationale for its further investigation as a therapeutic target for pulmonary TB.