Macrophage differential genes and their functions in silicosis based on single cell RNA sequencing and spatial transcriptome sequencing
雄性C57BL/6小鼠（5~6周龄，22~30 g）随机分为4组，即生理盐水（NS）组7 d、NS组56 d、SiO2组7 d及SiO2组56 d，每组各1只。SiO2组小鼠通过气管滴注SiO2悬液（0.2 g·kg−1，50 mg·mL−1）构建矽肺模型，对照组小鼠给予相同体积NS，第7、56天分别摘取右肺用于scRNA-seq以及左肺用于空间转录组测序。利用主成分分析技术和均匀流形逼近和投影降维，捕获细胞群。应用R语言的Find Markers函数分析两组肺组织中巨噬细胞的DEGs变化，对相应的DEGs进行基因本体富集分析和京都基因与基因组百科全书信号通路分析，同时应用STRING及Cytoscape软件的CytoHubba插件进行蛋白相互作用网络分析，筛选出关键（Hub）基因。运用空间转录组测序探究Hub基因在肺组织切片上的原始位置以及在肺巨噬细胞中的映射。最后验证Hub基因在矽肺病患者肺组织和小鼠矽肺模型中表达水平的关联性以及运用受试者工作特征曲线验证Hub基因诊断效能。体外实验应用细胞活力检测技术，验证SiO2刺激下小鼠巨噬细胞（RAW264.7）活力的变化。
Ccl2、Ccl7、Ptgs2，scRNA-seq显示与NS组相比，它们在SiO2组的表达水平升高且聚集在新增的巨噬细胞中，空间转录组测序显示它们聚集在炎性伴有结节病灶区； CCL7、PTGS2在矽肺患者肺组织中表达量相较于健康者增高，受试者工作曲线下的面积分别为0.850和0.786。与0 h相比，SiO2刺激下3 h、6 h、12 h RAW264.7细胞活力增强（ P<0.05）。 结论
Ccl2、Ccl7、Ptgs2）和2个潜在诊断基因（ CCL7、PTGS2）可能是潜在的矽肺早期阶段的分子生物学标志物，对矽肺的发生发展和预后存在一定的影响。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.
To explore differentially expressed genes DEGs in silicosis macrophages by scRNA-seq combined with spatial transcriptome sequencing and analyze the potential diagnostic genes.
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 vitroexperiments 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 PTGS2expressions 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 ( CCL7and 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.