Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

Abstract

Neutrophil extracellular traps (NETs) that are produced in the tumour microenvironment (TME) have been suggested to play an essential role in the dissemination of metastatic cancer under multiple infectious and inflammatory conditions. However, the functions of NETs in promoting non-small cell lung cancer (NSCLC) metastasis and the underlying mechanisms remain incompletely understood. Here, we found that NETs promoted NSCLC cell invasion and migration by inducing epithelial to mesenchymal transition (EMT). To explore how NETs contribute to NSCLC metastasis, microarrays were performed to identify substantial numbers of long noncoding RNAs (lncRNAs) and mRNAs that were differentially expressed in NSCLC cells after stimulation with NETs. Interestingly, we observed that the expression of lncRNA MIR503HG was downregulated after NETs stimulation, and ectopic MIR503HG expression reversed the metastasis-promoting effect of NETs in vitro and in vivo. Notably, bioinformatics analysis revealed that differentially expressed genes were involved in the NOD-like receptor and NF-κB signalling pathways that are associated with inflammation. NETs facilitated EMT and thereby contributed to NSCLC metastasis by activating the NF-κB/NOD-like receptor protein 3 (NLRP3) signalling pathway. Further studies revealed that MIR503HG inhibited NETs-triggered NSCLC cell metastasis in an NF-κB/NLRP3-dependent manner, as overexpression of NF-κB or NLRP3 impaired the suppressive effect of MIR503HG on NETs-induced cancer cell metastasis. Together, these results show that NETs activate the NF-κB/NLRP3 pathway by downregulating MIR503HG expression to promote EMT and NSCLC metastasis. Targeting the formation of NETs may be a novel therapeutic strategy for treating NSCLC metastasis.

Keywords: MIR503HG; NOD-like receptor protein 3 (NLRP3); epithelial to mesenchymal transition (EMT); neutrophil extracellular traps (NETs); non-small cell lung cancer (NSCLC).

Figure 1

The release of neutrophil extracellular traps (NETs) by neutrophils promotes migration and invasion of NSCLC. (A) The morphology of neutrophils isolated from healthy donors’ blood was observed by Giemsa staining (magnification, 1000×). (B) The neutrophil viability was assessed by trypan blue dye exclusion assays (magnification, 100×). (C) Representative images and quantification of NETs formation of neutrophils from healthy donors (HD) and NSCLC patients. MPO (red), cit-H3 (green), and DAPI (blue), respectively (magnification, 50×; scale bar, 200μm). (D) Representative images of NETs formation in NSCLC patients’ normal lung tissues and tumor tissues that were detected by con-focal microscopy. cit-H3 (red), Ly6g (green), and DAPI (blue), respectively (magnification, 200×; scale bar, 100µm and magnification, 400×; scale bar, 50µm). (E) Representative images of PMA-induced NETs formation of neutrophils from HD stained with MPO and cit-H3 were detected by immunofluorescence microscope; MPO (red), cit-H3 (green), and DAPI (blue), respectively (magnification, 50×; scale bar, 200μm). Transwell invasion (F) and wound healing assays (G) were performed to identify the effects of NETs on A549 and SK-MES-1 cells invasion (magnification, 100×) and migration (magnification, 50×). (H) Western blot analyzing the expressions levels of EMT markers protein (N-cadherin, E-cadherin, and Vimentin) in A549 and SK-MES-1 cells treated with NETs. (^*P < 0.05, ^**P < 0.01).

Figure 2

MIR503HG is downregulated in NSCLC cells with NETs stimulation and is associated with poor survival of NSCLC. (A) Volcano plot illustrating the differentially expressed lncRNAs in A549 cells treated with or without NETs for 12 h (|log₂ fold change (FC)| > 2, P-value < 0.01). (B) Heat map showing the top 30 differentially down-regulated lncRNAs in A549 cells after treatment with NETs for 12 h. Red means up-regulated, blue means down-regulated, separately. (C) Relative expression of MIR503HG in A549 and SK-MES-1 cells with or without NETs stimulation. (D) MIR503HG is expressed at a lower level in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumor compared to corresponding adjacent normal lung tissues according to the TCGA database. (E) The expression of MIR503HG in 50 paired NSCLC tumors and normal tissues were quantified by qRT-PCR. (F) Kaplan-Meier analysis of metastasis risk of 50 NSCLC patients divided into two groups based on a middle cutoff of MIR503HG expression. (G) MIR503HG is mainly located in the nuclear of NSCLC cells. U6 snRNA (nuclear reserved) and GAPDH mRNAs (exposed to cytoplasm) were used as controls. Data are mean ± SD (n=3). (^**P < 0.01).

Figure 3

Overexpression of MIR503HG substantially reversed the metastasis-promoting effect of NETs on NSCLC in vitro and vivo. (A) Expression of MIR503HG was successfully up-regulated in A549 and SK-MES-1 cells. (B, C) Wound healing (magnification, 50×) and invasion assays (magnification, 100×) of NSCLC cells that stable transfection of MIR503HG vector versus control vector both treat with NETs 12 h. (D) Western blot analysis of the expression of EMT (N-cadherin, E-cadherin, Vimentin) in control and MIR503HG overexpressing A549 and SK-MES-1 cells after NETs treated 12 h. (E) Schematic diagram showing the experimental design of the effect of MIR503HG on NETs-induced metastasis. Representative images of the gross lung (F) and H&E staining (G) of metastatic lung nodules in mice specimens. (H) Quantification of the number, volume, and maximum size of metastatic lung nodules. Data are mean ± SD (n=5 nude mice in each group). (magnification, 100×; scale bar, 200 μm, magnification, 200×; scale bar, 50 μm). (I) Immunohistochemistry (IHC) detection of N-cadherin, E-cadherin, and Vimentin revealed EMT formation in the NETs-induced lung metastasis model (magnification, 100× and 400×). (^**P < 0.01).

Figure 4

NETs promote migration and invasion of NSCLC by activating the NLRP3 inflammasome. (A) Up-regulated NSCLC-related pathways in response to NETs stimulated revealed by KEGG enrichment. (B) The mRNA expression of NLRP3, Caspase1, IL-1β and IL-18 in A549 cells was analyzed by qRT-PCR after NETs stimulation at different times. (C) Western blotting was used to analyze the expression of NLRP3 and Caspase1 in A549 and SK-MES-1 cells after NETs stimulation for different periods. (D) Immunofluorescence was used to observe the expression of ROS in A549 and SK-MES-1 cells treated with NETs for 12 h (magnification, 100×). The expression of NLRP3, Caspase1, IL-1β and IL-18 were detected by qRT-PCR (E) and Western blot (F) in A549 and SK-MES-1 cells after treating with NETs and NLRP3 inflammasome inhibitor MCC950, respectively. (G–I) Estimate the effect of NETs on the level of N-cadherin, E-cadherin, and Vimentin (Western blot) (G) in A549 and SK-MES-1 cells and the capacity of invasion (transwell invasion assays; magnification, 100×) (H), migration (wound healing assays; magnification, 50×) (I) when inhibiting the expression of the NLRP3 inflammasome by MCC950. (^*P < 0.05, ^**P < 0.01).

Figure 5

NLRP3 inflammasome mediated the effect of MIR503HG to inhibit NETs-triggered metastasis of NSCLC. (A, B) The protein and mRNA expression of NLRP3 and Caspase1 in A549 and SK-MES-1 cells with MIR503HG overexpression were analyzed by Western blot (A) and qRT-PCR (B) after NETs were stimulated. (C) A negative relationship between MIR503HG and NLRP3 in NETs-induced NSCLC cells is presented by correlation analysis. (D) Overexpression of NLRP3 attenuated the effect of MIR503HG in inhibiting NETs-triggered EMT in NSCLC cells by Western blot. (E, F) Overexpression of NLRP3 effectively reverses the effect of MIR503HG in inhibiting NETs-triggered promotion of NSCLC cells metastasis using transwell assay (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F). (^*P < 0.05, ^**P < 0.01).

Figure 6

NLRP3 inflammasome induced by NETs promotes NSCLC progression is associated with the activation of NF-κB. (A) The protein expression level of p-p50, p50, p-p65, p65 in NSCLC cells treated with NETs was detected by Western blotting. (B) Nuclear translocation of NF-κB in A549 and SK-MES-1 cells treated with NETs or combined with DNase I were detected by con-focal microscopy (magnification, 3000×; scale bar, 5μm). Immunofluorescence assays (C) and Western blot (D) were used to detect the effect of NETs on NLRP3 inflammasome in A549 and SK-MES-1 cells after p50 knockdown (magnification, 200×; scale bar, 50 μm). (E) Downregulation of p50 attenuated the effect on promoting EMT of NETs in NSCLC cells by Western blot. (F, G) Downregulated p50 reverses NETs-induced promotion of NSCLC cells metastasis using transwell assay (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F).

Figure 7

MIR503HG inhibits NETs-triggered NSCLC cells metastasis capacity and NLRP3 inflammasome activation dependently on NF-κB. (A) Western blotting was used to detect the changes of p-p50, p50, p-p65 and p65 in MIR503HG overexpression A549 and SK-MES-1 cells after NETs treatment. (B) qRT-PCR and (C) Western blotting analyses of up-regulating p50 in A549 and SK-MES-1 cells. (D) Analysis of the NLRP3 and Caspase1 protein levels in MIR503HG-overexpressed NSCLC cells transfected with p50-pcDNA3.1 and pcDNA3.1 vector by western blot with NETs stimulated. Transwell invasion (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) were performed to identify the effects of NETs on MIR503HG-overexpressed NSCLC cells invasion and migration transfected with p50-pcDNA3.1 and pcDNA3.1 vector. (^*P < 0.05, ^**P < 0.01).