Single-cell RNA-Seq analysis of molecular changes during radiation-induced skin injury: the involvement of Nur77

Abstract

Introduction: Ionizing radiation has been widely used in industry, medicine, military and agriculture. Radiation-induced skin injury is a significant concern in the context of radiotherapy and accidental exposure to radiation. The molecular changes at the single-cell level and intercellular communications during radiation-induced skin injury are not well understood. Methods: This study aims to illustrate this information in a murine model and human skin samples from a radiation accident using single-cell RNA sequencing (scRNA-Seq). We further characterize the functional significance of key molecule, which may provide a potential therapeutic target. ScRNA-Seq was performed on skin samples from a nuclear accident patient and rats exposed to ionizing radiation. Bioinformatic tools were used to analyze the cellular heterogeneity and preferential mRNAs. Comparative analysis was performed to identify dysregulated pathways, regulators, and ligand-receptor interactions in fibroblasts. The function of key molecule was validated in skin cells and in three mouse models of radiation-induced skin injury. Results: 11 clusters in human skin and 13 clusters of cells in rat skin were depicted respectively. Exposure to ionizing radiation caused changes in the cellular population (upregulation of fibroblasts and endothelial cells, downregulation of keratinocytes). Fibroblasts and keratinocytes possessed the most interaction pairs with other cell lineages. Among the five DEGs common to human and rat skins, Nur77 was highly expressed in fibroblasts, which mediated radiosensitivity by cell apoptosis and modulated crosstalk between macrophages, keratinocytes and endothelial cells in radiation-induced skin injury. In animal models, Nur77 knock-out mice (Nur77 ^-/-) showed more severe injury after radiation exposure than wild-type counterparts in three models of radiation-induced skin injury with complex mechanisms. Conclusion: The study reveals a single-cell transcriptional framework during radiation-induced skin injury, which provides a useful resource to uncover key events in its progression. Nur77 is a novel target in radiation-induced skin injury, which provides a potential therapeutic strategy against this disease.

Keywords: ionizing radiation; orphan nuclear receptor 77 (Nur77); radiation-induced skin injury; single-cell RNA sequencing (scRNA-Seq); skin.

Figure 1

Cell type identification by scRNA-Seq analysis in radiation-induced skin injury of rats. (A) Schematic of the workflow showing the overall strategy of scRNA-Seq to create a rat mononuclear cell atlas and subjected to droplet-based 10x Genomics. (B) The t-SNE plot displays cell clusters with combined groups. Each dot represents a single cell. (C) Dot plot showing the expression of representative genes for each cell type. (D) The t-SNE plot displays main cell types in each group of skin tissues. (E) Bar plots show the proportions that each group contributes to each cluster. (F) Heatmap showing gene expression signatures of each cell type. (G) An overview of cell-cell interactions. Arrow and edge color indicate direction Circle network plots showing weights/strengths of cell-cell interactions generated with CellChat in different groups.

Figure 2

Cutaneous cell type identification by scRNA-Seq analysis of a human patient in a radiation accident. (A) Schematic of the workflow showing the overall strategy of scRNA-Seq to create a human mononuclear cell atlas and subjected to droplet-based 10x Genomics. (B) The t-SNE plot displays human skin cell clusters. Each dot represents only one cell. (C) The t-SNE plot displays human skin cell types with or without radiation. (D) Dot plot showing the expression of representative genes for each cell type. (E) The t-SNE plot displays human skins cell types with or without radiation. (F) Bar plots show the proportions that each group contributes to each cluster. (G) Circle network plots showing number (left) and weights/strengths of cell-cell interactions generated with CellChat. (H) Top five differential expressed genes (DEGs) with Fold change (FC) values. (I) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses of DEGs (differentially expressed genes). Bubble plot of KEGG for up-regulated and down-regulated DEGs.

Figure 3

Changes in the transcriptional profiles of skin fibroblast during radiation-induced skin injury. (A) Bubble plot of TFs alterations in rat and human skin cells with or without radiation. (B) Venn plots showing the number of shared upregulated (upper) and downregulated (lower) DEGs between different groups of human and rat skin samples. Violin plots showing the expression levels of 5 common DEGs in human and rat skin. (C) The t-SNE plot displays rat (left) and human (right) fibroblast. Bar plots showing the cell number of each cell subtypes contributed. (D) Violin plot showing Nur77 gene expression changes across different fibroblast subcluster in rat (left) and human (right). (E) Pseudotime ordering on rat fibroblasts and human fibroblasts (F) arranged them into a major trajectory, with two minor bifurcations. Each dot represents a single cell. The black arrow indicates the start and direction of the trajectory. Feature plots of expression distribution for Nur77 across pseudotime. (G) Heatmap showing the top 5 markers for fibroblast subcluster from the rat (left) and human (right) skin.

Figure 4

Nur77 is involved in the irradiation process of skin cells. (A) qRT-PCR analysis of Nur77 mRNA expression in response to radiation in WS1 and HaCaT cells. (B) Western blotting analysis showing Nur77 expression in WS1 and HaCaT cells after different time post irradiation or different dose of irradiation. (C) and (D) Detection of radiation-induced nuclear-cytoplasmic distribution of Nur77 determined by preforming immunofluorescence analysis separating nuclear and cytoplasmic fraction and separating nuclear and cytoplasmic fractions. (E) Expression of Nur77 in normal and irradiated human skin tissues. (F) Western blot analysis showing the distribution of Nur77 in different organs and changes over time from 3 to 6 days after irradiation. (G) The effect of C-DIM8 on ROS production after different dose of irradiation as determined by DCFH-DA staining in WS1 and HaCaT cells. (H) The effect of C-DIM8 on cell apoptosis determined by AV/PI staining in WS1 and HaCaT cells. (I) The effect of Nur77 inhibitor C-DIM8 on radiosensitivity as determined by a colony formation assay following different doses of radiation. (J) Western blotting analysis showing cell death-related biomarker expression in irradiated WS1 cells treated with C-DIM8. *P < 0.05 and **P < 0.01, compared with the control group. Scale bar = 200 μm.

Figure 5

Loss of Nur77 aggravates radiation-induced skin injury in mouse models. (A) The phenotypes and genotypes of wild-type (Nur77^+/+) and Nur77 knockout (Nur77^-/-) mice. (B) Schematic of the workflow showing the establishment of the three mouse models with radiation-induced skin injury. 1) Acute radiation-induced skin injury; 2) Radiation fractionation; 3) A mouse model of full-thickness skin wounds combined with 4 Gy total-body irradiation. Loss of Nur77 aggravates radiation-induced skin injury in three mouse models. (C) Pictures showing the weight change and the scoring curves of the whole course of radiogenic injury in mice with different Nur77 genotypes. (D) Wound healing, body weight, and wound score results of radiation fractionation model in wild-type and Nur77 knockout mice. (E) Wound healing, body weight, and wound healing score results of the radiation combined injury model in mice. *P < 0.05 and **P < 0.01, compared with the control group.

Figure 6

scRNA-Seq reveals the complex mechanism by which Nur77 mediates radiation-induced skin injury. (A) Diagram displaying the process of sequencing single cells from radiation-induced skin injury samples obtained from wild-type (Nur77^+/+) and Nur77 knockout (Nur77^-/-) mice. (B) The t-SNE plot displays main cell types in wild-type and Nur77 knockout mice. Each dot represents only one cell. (C) Dot plot showing the expression of representative genes for each cell type. (D) The U-MAP plot displays cell types mouse skin with or without radiation. Each dot represents only one cell. (E) Bar plots show the proportions that each group contributes to each cluster. (F) The Venn diagram shows the number of up-regulated DEpcGs and down-regulated DEpcGs in different cell types. (G) Significant signaling pathways were ranked based on differences in the overall information flow within the inferred networks between Nur77^-/- and Nur77^+/+ mouse skin. The overall information flow of a signaling network is calculated by summarizing all communication probabilities in that network. An overview of cell-cell interactions. Arrow and edge color indicate direction. Bar plots showing overall information flow of each signaling pathway. (H) Heatmap shows outgoing signaling patterns of Nur77^-/- and Nur77^+/+ mouse skin. (I) Comparison of the significant ligand-receptor pairs between Nur77^-/- and Nur77^+/+ mouse skin, which contribute to the signaling from fibroblast to other cells.