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NOS2/miR-493-5p Signaling Regulates in the LPS-Induced Inflammatory Response in the RAW264.7 Cells

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Abstract

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.

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Data Availability

The authors declare that all relevant data that support the findings of this study are available within the article. Additional data are available from the corresponding author upon reasonable request.

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Author notes

  1. Xiaofei Li and Yongrui Yang have contributed equally to this work.

Authors and Affiliations

  1. Clinical Laboratory, The Third People’s Hospital of Kunming, Kunming, China

    Xiaofei Li & Feng Luo

  2. Department of Hepatology, The Third People’s Hospital of Kunming, Kunming, China

    Yongrui Yang

  3. Department of Respiratory Medicine, The Third People’s Hospital of Kunming, Kunming, China

    Nihong Lu

  4. School of Public Health, Dali University, Dali, China

    Ru Fan

  5. School of Life Science and Technology and School of Mechanical Engineering, Xi’an Jiaotong University, Xi’an, 710049, China

    Niancai Peng

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  1. Xiaofei Li
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XL and NP designed the experiments and conceived the study; XL, YY, and NL performed the experiments; FL and RF performed the statistical analysis; all the authors approved the manuscript for publication.

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Correspondence to Niancai Peng.

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Supplementary Information

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10528_2022_10297_MOESM1_ESM.tif

Supplementary file1 (Functional enrichment analysis of DEGs. A-C The genes were divided into three categories: biological process (A), molecular function (B), and cellular component (C). D The genes were enriched in different KEGG pathways.)

10528_2022_10297_MOESM2_ESM.tif

Supplementary file2 (Effects of miR-493-5p overexpression/inhibition on the NOS2 expression in RAW264.7 cells treated with LPS. A The RAW264.7 cells were treated with miR-493-5p inhibitors for 24 h and were followed by 100 ng/ml LPS for 24 h, the mRNA expression level of NOS2 was determined by qRT-PCR. (B) (A) The RAW264.7 cells were treated with miR-493-5p mimics for 24 h and were followed by 100 ng/ml LPS for 24 h, the mRNA expression level of NOS2 was determined by qRT-PCR. N = 3. Significant differences between groups were illustrated as *P<0.05.)

10528_2022_10297_MOESM3_ESM.tif

Supplementary file3 (Effects of miR-493-5p overexpression or inhibition on inflammatory responses in RAW264.7 cells. (A–F) The RAW264.7 cells were treated with miR-493-5p mimics, miR-493-5p inhibitors, mimics NC or inhibitors NC for 24 h, the mRNA expression levels of IL-1β (A), IL-6 (B), and TNF-α (C) were determined by qPCR; the protein expression levels of IL-1β (D), IL-6 (E), and TNF-α (F) were determined by ELISA assay. N = 3. Significant differences between groups were illustrated as *P<0.05 and **P<0.01.)

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Li, X., Yang, Y., Lu, N. et al. NOS2/miR-493-5p Signaling Regulates in the LPS-Induced Inflammatory Response in the RAW264.7 Cells. Biochem Genet 61, 1097–1112 (2023). https://doi.org/10.1007/s10528-022-10297-2

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