Mechanism Of Neutrophil Extracellular Traps In Promoting Non-small Cell Lung Cancer Metastasis And Radiotherapy Resistance

Part 1:Effect and Mechanistic Study of the Regulation of IncRNA MIR503HG Activating the NF-κB/NLRP3 Pathway to Promote Non-Small Cell Lung Cancer Metastasis by Neutrophil Extracellular TrapsObjective:Tumor metastasis is a major cause of the high incidence and mortality rate of cancer and is also the main reason for the failure of cancer treatment.However,current medical treatments are still limited against tumor metastasis,and the treatment effects are unsatisfactory.Therefore,further elucidation of the molecular mechanism of non-small cell lung cancer(NSCLC)metastasis has great significance for the prevention and treatment of NSCLC.Current research suggests that neutrophil extracellular traps(NETs)are associated with NSCLC metastasis.However,the specific mechanism is still unclear,this study will explore the specific mechanism by which NETs promote NSCLC metastasis.Microarrays were used to screen the key expression profiles of lncRNAs and related signaling pathways involved in NETs promoting NSCLC metastasis.This study aims to clarify the effect and specific mechanism of MIR503HG and its downstream signaling pathway in NETs promoting NSCLC metastasis in vitro and in vivo experiments.Methods:Detection of the expression of NETs in peripheral blood of healthy individuals and NSCLC patients;detection expression of NETs in NSCLC tumor tissue and adjacent tissue.Extraction and identification of NETs,stimulation of NSCLC cells with NETs,and invasion,migration,and epithelial-mesenchymal transition(EMT)of NSCLC cells was assessed. RNA samples from NSCLC cells stimulated with NETs were collected and sent for high-throughput detection of transcriptome RNA microarray.Bioinformatics analysis was used to screen for differentially expressed IncRNAs and mRNAs molecules and signaling pathways enriched with differentially expressed genes related to NETs promoting metastasis,and key lncRNAs and downstream signaling pathways involved in NETs promoting metastasis were identified.qRT-PCR was employed to validate the transcriptome microarray results,and lncRNA MIR503HG basic information was obtained from online databases such as UCSC and NCBI;the protein-coding ability of MIR503HG was analyzed by CPC 2.0 and CPAT online databases,and the subcellular localization of MIR503HG was predicted by the lncATLAS online database.The subcellular localization of MIR503HG was verified by nuclear-cytoplasmic separation technology.The GEPIA online database was used to analyze the expression of MIR503HG in NSCLC tumor tissue and adjacent tissue of patients;clinical samples were used to verify the expression of MIR503HG and analyze the risk of metastasis in NSCLC patients.Overexpressing MIR503HG NSCLC cell lines were constructed,and the effect of NETs on invasion,migration,and EMT of overexpressing MIR503HG NSCLC cells was detected.The pulmonary metastasis xenograft model was adopted to evaluate the effect of NETs on overexpressing MIR503HG NSCLC cells metastasis in vivo.The effect of NETs on NF-κB/NLRP3 inflammasome signaling pathway and reactive oxygen species(ROS)expression was detected in NSCLC cells.The key molecules of the NF-κB/NLRP3 inflammasome signaling pathway were silenced by siRNA interference technology and NLRP3 inhibitor MMC950,and the effect of NETs on invasion,migration,and EMT of NSCLC cells was detected.The effects of NETs on NF-κB/NLRP3 inflammasome signaling pathway in NSCLC cells with overexpression of MIR503HG was detected,and the correlation between the expression of MIR503HG and NLRP3 was analyzed.Transfecting NLRP3 plasmids into NSCLC cells with overexpression of MIR503HG,detecting the effect of NETs on invasion,migration,and EMT of NSCLC cell,and detecting the role of NF-κB/NLRP3 inflammasome in NETs promoting NSCLC metastasis by inhibiting the expression of MIR503HG by the rescue experiment.RNA pull-down assay and mass spectrometry(MS)was performed to identify MIR503HG-associated proteins that might be involved in regulating the biological processes of NLRP3 transcription.Screening the candidate key protein by siRNA and further validating the relationship between the key protein(C/EBP β)and MIR503HG by RNA pull-down and RIP assay.The relationship between C/EBP β expression and NSCLC prognosis was analyzed by GEPIA online database.The correlation between C/EBP β and NLRP3 expression was analyzed by GEPIA online database.The expression of NLRP3 and the invasion and migration capacities of NSCLC cells were determined after transfecting C/EBP β plasmids.Results:1.Neutrophils from NSCLC patients’ peripheral blood produce spontaneously more NETs than healthy individuals.More NETs are expressed in tumor tissues of NSCLC patients compared to adjacent normal tissues.2.Phorbol myristate acetate(PMA),an inducer of NET,can induce the formation of NETs in neutrophils.3.NETs can promote the invasion,migration and EMT of NSCLC cells(A549,SK-MES-1).4.Substantial differences in lncRNA and mRNA were confirmed after stimulating A549 cells with NETs by differential expression analysis,and the expression of MIR503HG was down-regulated.qRT-PCR validation results were consistent with the transcriptome microarray results.5.MIR503HG is located at position 3 of the X chromosome long arm 2 region 6 band(Xq26.3),with a length of 786bp.Predictive results show that MIR503HG have no protein-coding ability and is mainly expressed in the nucleus.Nuclear-cytoplasmic fractionation experiments confirmed that MIR503HG is mainly located in the nucleus.MIR503HG was lower expressed in NSCLC tumor tissues by analyzing TCGA NSCLC datasets and clinical specimen.6.Overexpression of MIR503HG can inhibit the invasion and migration ability of NSCLC cells promoted by NETs and suppress EMT in vitro experiments.Overexpression of MIR503HG can inhibit the formation of lung metastasis xenograft model promoted by NETs in vivo experiments.7.Transcriptome microarray of differential mRNA expression showed that NETs can activate the NF-κB/NLRP3 inflammasome signaling pathway in NSCLC cells.NLRP3 and Caspasel expression was highest at 12h after stimulation,and NETs can induce ROS generation.Inhibition of the NF-κB/NLRP3 inflammasome signaling pathway can inhibit the effect of NETs on NSCLC cell invasion,migration ability,and EMT.8.Overexpression of MIR503HG can inhibit the activation of the NF-κB/NLRP3 inflammasome by NETs.MIR503HG expression is negatively correlated with NLRP3 expression.9.Transfection of NLRP3 plasmids in NSCLC cells overexpressing MIR503HG can reverse the inhibitory effect of MIR503HG on NETs promotion of NSCLC cell invasion,migration,and EMT.10.RNA pull-down and protein spectrum detection found that the sense strand of MIR503HG can specifically bind to 326 molecular proteins.According to the prediction of the Human TFDB website,10 proteins may bind to the promoter of NLRP3,promoting the process of transcriptional regulation of NLRP3.RNA pull-down and RIP experiments confirmed that C/EBP β specifically binds to MIR503HG.11.Online database GEPIA analysis found that high expression of the C/EBP βmolecule is associated with poor overall survival(OS)and disease-free survival(DFS)prognosis in NSCLC patients.12.Online database GEPIA analysis found that C/EBP β and NLRP3 are positively correlated in NSCLC patients.Transfection of C/EBP β plasmids in NSCLC cells can upregulate the expression of NLRP3 and promote NSCLC cell invasion and migration.Conclusion:NETs can promote NSCLC metastasis by inhibiting the expression of MIR503HG and further activating the NLRP3 inflammasome pathway by regulating C/EBP β.The "NETs-MIR503HG-C/EBPβ-NF-κB/NLRP3" signal axis plays an important role in promoting NSCLC metastasis.Part Two: Study on the Role and Preliminary Mechanisms of Radiotherapy-Induced Extracellular Vesicles(EVs)from Non-Small Cell Lung Cancer Tissue in Inducing Neutrophil Extracellular Traps(NETs)and Promoting Radiotherapy ResistanceObjective: Radiotherapy is one of the most important treatments for non-small cell lung cancer(NSCLC).However,the development of radioresistance in tumor cells to ionizing radiation(IR)is the main reason for radiotherapy failure and a crucial factor contributing to a poor prognosis in patients.Therefore,a further understanding of the molecular mechanisms of radioresistance in NSCLC is of great significance for improving the efficacy of radiotherapy.Neutrophil extracellular traps(NETs)are associated with radioresistance.Similarly,radiotherapy can induce the release of extracellular vesicles(EVs)from the tumor microenvironment and alter the molecular content of EVs.This study aims to explore the role of NETs in NSCLC radiotherapy resistance,extract EVs from NSCLC tissue after radiotherapy,explore the function and effects of EVs in NSCLC radiotherapy,and elucidate the impact of EVs on the release of NETs from neutrophils.Methods: A subcutaneous NSCLC model was established using LLC cells in C57BL/6 mice,and different radiation doses were administered.Immunofluorescence was used to detect the expression of NETs in the tumors.NETs inhibitor DNase I was injected intraperitoneally to degrade NETs,and the effect of radiotherapy on the proliferation of subcutaneous tumors was observed after NETs inhibition.EVs were extracted from tumor tissues that received radiotherapy(IR-EVs)or did not receive radiotherapy(NC-EVs),and the morphological structure of EVs was observed using transmission electron microscopy(TEM).EV markers were detected using Western blot analysis.The Di R-labeled EVs were injected into the tail vein.The uptake of EVs by tumor cells was confirmed by bioluminescence imaging technology,and the effect of EVs on tumor tissue radiotherapy was observed.Neutrophils were extracted from mouse bone marrow,and stimulated with PKH26-labeled EVs for 4 hours,the uptake of EVs by neutrophils and the expression of NETs after EVs stimulation were detected.Clone formation assay was used to detect the effect of EV-induced NETs formation on the radiosensitivity of LLC cells,and Transwell was used to detect the invasive and migration abilities of LLC cells.EVs were injected into the mice via the tail vein,and the expression of NETs in the lungs of mice was detected to construct a mouse lung metastatic tumor model,and the metastatic tumor formation of LLC cells in the lungs was observed.Results: 1.Radiotherapy can induce the formation of NETs in mouse NSCLC tumor tissues in a dose-dependent manner.2.Administration of the NETs inhibitor DNase I by intraperitoneal injection can increase the radiosensitivity of LLC tumor cells in mice.3.EVs were isolated and extracted from LLC tumor tissue,and transmission electron microscopy showed that the EVs were typical membrane-bound vesicles with circular,elliptical,and cup-shaped structures,with a diameter of approximately 100 nm.Western blot results showed that the EV-specific markers CD63 and Tsg101 were positive,while the GM130 and Calnexin proteins were negative.4.Bioluminescence imaging technology confirmed that EVs can be taken up by LLC tumor cells in mice through tail vein injection and can promote the proliferation of tumor cells after radiotherapy.5.Confocal microscopy confirmed that neutrophils can take up PKH26-labeled EVs after co-culture in vitro.6.Compared with NC-EVs,IR-EVs significantly increased the formation of NETs in a dose-dependent manner.7.IR-EVs can promote LLC cell radiation resistance by inducing NETs,as well as promoting LLC cell invasion and migration.8.IR-EVs can induce the formation of NETs in the lungs of mice in vivo and promote the formation of metastatic tumors of LLC cells in the lungs.Conclusion: Radiotherapy can induce the formation of NETs in NSCLC tumors and promote radiotherapy resistance.EVs from NSCLC tissues that have undergone radiotherapy can induce the formation of NETs in neutrophils and promote the proliferation of tumor cells after radiotherapy.