Inhibition of EZH2 Ameliorates Sepsis Acute Lung Injury (SALI) and Non-Small-Cell Lung Cancer (NSCLC) Proliferation through the PD-L1 Pathway

Abstract

(1) Background: Both sepsis acute lung injury (SALI) and non-small-cell lung cancer (NSCLC) are life-threatening diseases caused by immune response disorders and inflammation, but the underlining linking mechanisms are still not clear. This study aimed to detect the shared gene signature and potential molecular process between SALI and NSCLC. (2) Methods: RNA sequences and patient information on sepsis and NSCLC were acquired from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was used to build a co-expression network associated with sepsis and NSCLC. Protein-protein interaction (PPI) analysis of shared genes was intuitively performed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The involvement of EZH2 in the tumor immune microenvironment (TIME) and sepsis immune microenvironment (IME) was assessed by R software. Western blot, flow cytometry, and other in vitro assays were performed to further confirm the function and mechanism of EZH2 in NSCLC and SALI. (3) Results: WGCNA recognized three major modules for sepsis and two major modules for NSCLC, and there were seven shared genes identified for the two diseases. Additionally, the hub gene EZH2 was screened out. It was shown that EZH2 was closely related to the IME in the two diseases. In the validation assay, our data showed that EZH2 was expressed at a higher level in peripheral blood mononuclear cells (PBMCs) of septic patients than those of healthy donors (HDs), and EZH2 was also expressed at a higher level in lipopolysaccharide (LPS)-induced PBMCs and non-small cell lung cancer (A549) cells. EZH2 inhibitor (GSK343) downregulated the proliferation ability of A549 cells in a concentration-dependent manner, parallel with the decreased expression level of PD-L1. Similarly, GSK343 inhibited PD-L1 protein expression and downregulated the level of proinflammatory factors in LPS-induced PBMCs. In the co-culture system of PBMCs and human type II alveolar epithelial cells (ATIIs), the addition of GSK343 to PBMCs significantly downregulated the apoptosis of LPS-induced ATIIs. (4) Conclusions: This study illustrated that EZH2 inhibition could ameliorate A549 cell proliferation and LPS-induced ATII apoptosis in parallel with downregulation of PD-L1 protein expression, which provided new insights into molecular signaling networks involved in the pathogenetics of SALI and NSCLC.

Keywords: EZH2; NSCLC; SALI; WGCNA; apoptosis.

Figure 1

Identification of modules linked to clinical features of SALI and NSCLC. (A,B) Cluster dendrogram of co-expressed genes in SALI (A) and NSCLC (B). (C,D) Heat map of module–trait relationships in SALI (C) and NSCLC (D). (E) Venn diagram of the shared genes between the SALI modules and NSCLC modules. (F) PPI network of shared genes. (G) GSEA of the top 6 enriched pathways in sepsis patients with high EZH2 expression. GSEA, Gene Set Enrichment Analysis.

Figure 1

Identification of modules linked to clinical features of SALI and NSCLC. (A,B) Cluster dendrogram of co-expressed genes in SALI (A) and NSCLC (B). (C,D) Heat map of module–trait relationships in SALI (C) and NSCLC (D). (E) Venn diagram of the shared genes between the SALI modules and NSCLC modules. (F) PPI network of shared genes. (G) GSEA of the top 6 enriched pathways in sepsis patients with high EZH2 expression. GSEA, Gene Set Enrichment Analysis.

Figure 2

EZH2 could be a potential biomarker for SALI and NSCLC. (A,B) Western blot assay was performed to assess the EZH2 expression level in healthy donors (HDs) and septic patients. The fold expression was measured by densitometric analysis. (C,D) Representative IHC staining of EZH2 in normal and NSCLC tissues from the HPA database. (E) ROC curve of EZH2 in NSCLC patients from the TCGA database. (F,G) Western blot assay was performed to assess the EZH2 expression level in LPS-induced PBMCs and A549. The fold expression was measured by densitometric analysis. Values are expressed as mean ± SD. ^#### p < 0.001, relative to the control group, n = 3.

Figure 2

EZH2 could be a potential biomarker for SALI and NSCLC. (A,B) Western blot assay was performed to assess the EZH2 expression level in healthy donors (HDs) and septic patients. The fold expression was measured by densitometric analysis. (C,D) Representative IHC staining of EZH2 in normal and NSCLC tissues from the HPA database. (E) ROC curve of EZH2 in NSCLC patients from the TCGA database. (F,G) Western blot assay was performed to assess the EZH2 expression level in LPS-induced PBMCs and A549. The fold expression was measured by densitometric analysis. Values are expressed as mean ± SD. ^#### p < 0.001, relative to the control group, n = 3.

Figure 3

Characterization of the shared gene EZH2 in immune cells. Comparison of immune cell subsets with high EZH2 expression and low EZH2 expression in sepsis patients’ PBMCs (A) and NSCLC patients’ lung tissues (B). Spearman correlation analysis of EZH2 and T cell exhaustion-related molecules in sepsis (C) and NSCLC (D). Markers include CD274, PDCD1, CTLA4, LAG3, GZMB, and PDCD1LG2 of T cell exhaustion. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ns: no significance.

Figure 4

GSK343 decreased the protein level of PD-L1 in LPS-induced PBMCs and A549. (A) The effect of 100 ng/mL of LPS on the expression of TNF-α in PBMCs. (B) The effect of 1 μg/mL of LPS on the expression of TNF-α in PBMCs. (C) Representative IHC staining of EZH2 in normal and NSCLC tissues from the HPA database. (D,E) Immunofluorescence was performed to detect the protein expression level of PD-L1. Magnification ×20, scale bar 50 μm. (F–H) The protein expression levels of PD-L1 in LPS-induced PBMCs and A549 were analyzed by Western blotting. The results were quantified by densitometry. The fold expression was measured by densitometric analysis. Data are presented as mean ± SD (n = 3 per group) of the representative data from three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ^# p < 0.05, ^## p < 0.01, ^### p < 0.005, ^#### p < 0.001, ns: no significance. The symbol (#) shows that the group is statistically different from the Con group.

Figure 4

GSK343 decreased the protein level of PD-L1 in LPS-induced PBMCs and A549. (A) The effect of 100 ng/mL of LPS on the expression of TNF-α in PBMCs. (B) The effect of 1 μg/mL of LPS on the expression of TNF-α in PBMCs. (C) Representative IHC staining of EZH2 in normal and NSCLC tissues from the HPA database. (D,E) Immunofluorescence was performed to detect the protein expression level of PD-L1. Magnification ×20, scale bar 50 μm. (F–H) The protein expression levels of PD-L1 in LPS-induced PBMCs and A549 were analyzed by Western blotting. The results were quantified by densitometry. The fold expression was measured by densitometric analysis. Data are presented as mean ± SD (n = 3 per group) of the representative data from three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ^# p < 0.05, ^## p < 0.01, ^### p < 0.005, ^#### p < 0.001, ns: no significance. The symbol (#) shows that the group is statistically different from the Con group.

Figure 5

GSK343 alleviated A549 proliferation. (A) Representative images of colony formation and cell migration. (B) Inhibition of EZH2 significantly reduced colony formation ability in a concentration-dependent way. Data are presented as mean ± SD (n = 3 per group) of the representative data from three independent experiments; *** p < 0.005, **** p < 0.001, ^#### p < 0.001, ns: no significance. The asterisk (*) demonstrates that the group is statistically different from the Con group.

Figure 6

GSK343 alleviated LPS-induced ATII apoptosis. (A,C) Flow cytometry was employed to detect the apoptosis rate of ATIIs. The cells were equally divided into the Con group, LPS group, GSK343 group, and LPS + GSK343 group. We evaluated the proportion of apoptotic cells (Q2 + Q3) in each group. (B) The effect of GSK343 on the expression of TNF-α in LPS-induced PBMCs. Data are presented as mean ± SD (n = 3 per group) of the representative data from three independent experiments; *** p < 0.005, **** p < 0.001, ns: no significance, ### p < 0.005, #### p < 0.001. The asterisk (*) shows that the group is statistically different from the Con group.

Figure 7

Overview of the interactions between SALI and NSCLC. The shared genes of SALI and NSCLC regulate PBMC and ATII apoptosis to enhance SALI and are involved in cancer-related pathways that promote NSCLC development. SALI, sepsis acute lung injury; NSCLC, non-small-cell lung cancer.