Sun Zhicheng, Pang Xiaoyang, Wang Xiyang, Zeng Hao
Department of Spinal Surgery, Xiangya Hospital of Central South University, Changsha, Hunan, China.
Department of Spinal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.
Front Microbiol. 2023 Aug 3;14:1236012. doi: 10.3389/fmicb.2023.1236012. eCollection 2023.
Macrophages play an important role in regulating the course of spinal tuberculosis within the bone microenvironment. This study aimed to investigate the differential expression of miRNA in macrophage-derived exosomes within the tuberculosis-infected bone microenvironment, to identify miRNAs that hold potential as diagnostic markers and therapeutic targets.
We established study cohorts for spinal tuberculosis, collected bone marrow blood samples, isolated macrophage exosomes, and performed exosome miRNA sequencing. A miRNA-mRNA co-expression network was constructed using WGCNA analysis. Gene GO analysis and KEGG pathway enrichment analysis were performed using KOBAS software. Target miRNAs were selected based on fold change, -value, and false discovery rate, and their validation was carried out using qRT-PCR and ROC curve studies. Subsequently, we constructed a target gene network for these miRNAs and performed KEGG pathway enrichment analysis to explore the potential signaling mechanisms involved in regulating the disease course of spinal tuberculosis.
Our findings revealed that macrophages from the tuberculosis-infected bone microenvironment exhibited an M1 phenotype. The successful extraction of exosomes from macrophage supernatants was confirmed through electron microscopy, particle size analysis, and protein blot analysis. Exosome miRNA-seq demonstrated that 28 miRNAs were up-regulated, while 34 miRNAs were down-regulated in individuals with spinal tuberculosis. GO analysis and KEGG pathway enrichment analysis indicated that the differentially expressed miRNAs were involved in various biological processes, cell components, molecular functions, and signaling pathways, which collectively contribute to the regulation of the disease course of spinal tuberculosis. Notably, miRNA-125b-5p was successfully selected based on fold change, -value, and false discovery rate. qRT-PCR validation further confirmed the significant up-regulation of miRNA-125b-5p in spinal tuberculosis. The ROC curve revealed that miR-125b-5p is a potential diagnostic biomarker for spinal tuberculosis. Moreover, construction of the miRNA-125b-5p target gene network and subsequent KEGG enrichment analysis highlighted the importance of MAPK, TNF, Ras, Rap1, and the PI3K-Akt signaling pathways in the regulation of the disease course of spinal tuberculosis.
Our study demonstrates differential expression of miRNAs in macrophage-derived exosomes in the tuberculosis-infected bone microenvironment. Specifically, MiRNA-125b-5p is significantly up-regulated in spinal tuberculosis and shows potential as a diagnostic biomarker for spinal tuberculosis.
巨噬细胞在骨微环境中调节脊柱结核病程方面发挥着重要作用。本研究旨在探讨结核感染骨微环境中巨噬细胞来源外泌体中miRNA的差异表达，以鉴定具有作为诊断标志物和治疗靶点潜力的miRNA。
我们建立了脊柱结核研究队列，采集骨髓血样本，分离巨噬细胞外泌体，并进行外泌体miRNA测序。使用WGCNA分析构建miRNA-mRNA共表达网络。使用KOBAS软件进行基因GO分析和KEGG通路富集分析。根据倍数变化、P值和错误发现率选择靶miRNA，并使用qRT-PCR和ROC曲线研究对其进行验证。随后，我们构建了这些miRNA的靶基因网络，并进行KEGG通路富集分析，以探索参与调节脊柱结核病程的潜在信号机制。
我们的研究结果显示，结核感染骨微环境中的巨噬细胞表现出M1表型。通过电子显微镜、粒径分析和蛋白质印迹分析证实了从巨噬细胞上清液中成功提取外泌体。外泌体miRNA测序表明，脊柱结核患者中有28种miRNA上调，34种miRNA下调。GO分析和KEGG通路富集分析表明，差异表达的miRNA参与了各种生物学过程、细胞成分、分子功能和信号通路，这些共同有助于调节脊柱结核的病程。值得注意的是，基于倍数变化、P值和错误发现率成功选择了miRNA-125b-5p。qRT-PCR验证进一步证实了miRNA-125b-5p在脊柱结核中显著上调。ROC曲线显示，miR-125b-5p是脊柱结核的潜在诊断生物标志物。此外，miRNA-125b-5p靶基因网络的构建及随后的KEGG富集分析突出了MAPK、TNF、Ras、Rap1和PI3K-Akt信号通路在调节脊柱结核病程中的重要性。
我们的研究证明了结核感染骨微环境中巨噬细胞来源外泌体中miRNA的差异表达。具体而言，miRNA-125b-5p在脊柱结核中显著上调，并显示出作为脊柱结核诊断生物标志物的潜力。
