Tuberculosis (TB) is a fatal infectious disease; however, the molecular mechanisms underlying the pathogenicity of TB remain elusive. The present study aims to identify potential biomarkers associated with Mycobacterium tuberculosis (M.tb) infection by using integrated bioinformatics and in vitro validation studies. GSE50050, GSE78706, and GSE108844 data from the gene expression omnibus (GEO) database were downloaded to identify differentially expressed genes (DEGs). The functions of DEGs were further subjected to gene ontology (GO) and KEGG pathway analysis. The hub genes from the DEGs were determined based on the protein–protein interaction (PPI) network analysis. Finally, the hub genes were experimentally validated using the in vitro functional studies. A total of 26 common DEGs were identified among GSE50050, GSE78706, and GSE108844. The functional enrichment analysis showed that the common DEGs were associated with cytokines response and TB pathways. The PPI network analysis identified nine hub genes. Further in vitro studies showed that nitric oxide synthase 2 (NOS2) was up-regulated in RAW264.7 cells upon lipopolysaccharides (LPS) stimulation, which was accompanied by increased inflammatory cytokines release. Furthermore, NOS2 was found to be a target of miR-493-5p, which was confirmed by luciferase reporter assay. NOS2 was repressed by miR-493-5p overexpression and was up-regulated after miR-493-5p inhibition in RAW264.7 cells. The rescue experiments showed that LPS-induced increase in the inflammatory cytokines of the RAW264.7 cells was significantly attenuated by NOS2 knockdown and miR-493-5p overexpression. Collectively, our results for the first time demonstrated that NOS2/miR-493-5p signaling pathway may potentially involve in the inflammatory response during bacterial infection such as M. tb infection.

结核病（TB）是一种致命的传染病；然而，结核病致病性的分子机制仍然难以捉摸。本研究旨在通过综合生物信息学和体外验证研究来确定与结核分枝杆菌( M.tb ) 感染相关的潜在生物标志物。下载来自基因表达综合 (GEO) 数据库的 GSE50050、GSE78706 和 GSE108844 数据，以识别差异表达基因 (DEG)。 DEGs的功能进一步进行基因本体（GO）和KEGG通路分析。 DEG 的中心基因是根据蛋白质-蛋白质相互作用 (PPI) 网络分析确定的。最后，利用体外功能研究对中心基因进行了实验验证。 GSE50050、GSE78706 和 GSE108844 中总共鉴定出 26 个常见 DEG。功能富集分析表明，常见的差异表达基因与细胞因子反应和结核通路相关。 PPI 网络分析确定了 9 个枢纽基因。进一步的体外研究表明，在脂多糖（LPS）刺激下，RAW264.7细胞中一氧化氮合酶2（NOS2）上调，并伴随着炎症细胞因子释放的增加。此外，NOS2被发现是miR-493-5p的靶标，这通过荧光素酶报告基因测定得到证实。在 RAW264.7 细胞中，NOS2 受到 miR-493-5p 过表达的抑制，并在 miR-493-5p 抑制后上调。拯救实验表明，NOS2 敲低和 miR-493-5p 过表达显着减弱了 LPS 诱导的 RAW264.7 细胞炎症细胞因子的增加。 总的来说，我们的结果首次证明NOS2/miR-493-5p信号通路可能参与结核分枝杆菌感染等细菌感染期间的炎症反应。