Macrophage differential genes and their functions in silicosis based on single cell RNA sequencing and spatial transcriptome sequencing
- VernacularTitle:基于单细胞RNA测序和空间转录组测序分析矽肺中巨噬细胞差异基因及其功能
- Author:
Xinbei ZHOU
1
,
2
;
Wei ZHANG
2
;
Jie CHAO
1
,
2
Author Information
- Publication Type:Selected article
- Keywords: silicosis; single cell RNA sequencing; spatial transcriptome sequencing; differentially expressed gene; protein-protein interaction network; enrichment analysis
- From: Journal of Environmental and Occupational Medicine 2022;39(12):1350-1358
- CountryChina
- Language:Chinese
- Abstract: Background The rise of single cell RNA sequencing (scRNA-seq) and spatial transcriptome sequencing technologies has allowed for intensive study of lung diseases, but both have been poorly studied in silicosis. Objective To explore differentially expressed genes DEGs in silicosis macrophages by scRNA-seq combined with spatial transcriptome sequencing and analyze the potential diagnostic genes. Methods Male C57BL/6 mice (5-6 weeks old, 22-30 g) were randomly divided into 4 groups: normal saline (NS) group for 7 d, NS group for 56 d, SiO2 group for 7 d, and SiO2 group for 56 d, with 1 mouse in each group. A silicosis model was constructed by tracheal drip injection of SiO2 suspension (0.2 g·kg−1, 50 g·cm−2), and the control mice were given the same volume of NS. The right lung was removed for scRNA-seq and the left lung for spatial transcriptome sequencing on day 7 and day 56, respectively. Cell populations were captured using principal component analysis techniques and dimensionality reduction of uniform manifold approximation and projection. The Find Markers function in R language was applied to analyze the DEGs changes of macrophages in two groups of lung tissues, and the corresponding DEGs were subjected to Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes signaling pathway analysis, while STRING and CytoHubba plug-ins of Cytoscape software were applied to protein-protein interaction network analysis to screen out key (Hub) genes. Spatial transcriptome sequencing was used to explore the original location of Hub genes on lung tissue sections and their mapping in lung macrophages. Finally, the correlation of Hub gene expression levels in lung tissues of silicosis patients and mouse silicosis models was verified, the diagnostic efficacy of Hub gene using subject operating characteristic curves (ROC). In vitro experiments by applying cell viability assay were conducted to verify the changes in viability of mouse macrophages (RAW264.7) under SiO2 stimulation. Results The scRNA-seq revealed a total of 20 clusters captured and defined. The results of scRNA-seq and spatial transcriptome sequencing showed an increased number of macrophages in the lung tissue of the SiO2 group compared to the NS group and clustered in the focal areas. Among the 97 macrophage DEGs screened out, 75 were up-regulated genes, and mainly enriched in chemotaxis and migration of neutrophils, chemokine receptor binding, tumor necrosis factor signaling pathway, cytokine-cytokine receptor interaction pathway, and interleukin-17 signaling pathway; and 22 were down-regulated genes, and mainly enriched in late endosomes, peroxisome proliferator-activated receptors signaling pathway, and alcoholic liver disease signaling pathway. A total of 2 core modules and 3 Hub genes were screened out, including Ccl2, Ccl7, and Ptgs2. The scRNA-seq showed that they were expressed at elevated levels in the SiO2 group compared to the NS group and clustered in additional macrophages, and the spatial transcriptome sequencing showed that they clustered in inflammatory areas with nodular lesions. The CCL7 and PTGS2 expressions were increased in the lung tissue of SiO2 patients compared with the healthy subjects, and the areas under the working curve of the subjects were 0.850 and 0.786, respectively. The viability of RAW264.7 cells was enhanced under SiO2 stimulation at 3 h, 6 h, and 12 h compared to those without the stimulation (P<0.05). Conclusion Bioinformatics screening have identified 3 Hub genes (Ccl2, Ccl7, and Ptgs2)and 2 potential diagnostic genes (CCL7 and PTGS2) in the lung tissue of silicosis mice, which may be potential molecular markers of early-stage silicosis with implications for the development and prognosis of silicosis.
