The Role Of α5-nicotinic Acetylcholine Receptor/NLRP3 Signaling Pathway In Lung Adenocarcinoma Cell Proliferation And Migration

BackgroundLung cancer has one of the worst prognose of all malignant tumors and the overall 5-year relative survival rate is 20%in most countries.Especially,non-small cell lung cancer(NSCLC)is a cancer that represents 85%of all lung cancer cases.The most important risk factor for lung cancer is still tobacco smoking,and the component of cigarettes which is called nicotine,induces the proliferation,migration and invasion of lung cancer cells by activating nicotinic acetylcholine receptors(nAChR).Genome wide association studies(GWAS)found that the locus on chromosome 15q24-26 encodes the α3,β4 and α5 subunits of the nAChRs,which lead to an increased risk of developing lung cancer,in particular,the CHRNA5 gene,which encodes a5-nAChR,is closely associated with lung cancer.nAChRs are ligand-gated ion channel proteins composed of homologous or heterologous pentamers.Different combinations of alpha(α1-α10)or nonalpha(β1-β4,γ,δ,or ε)types produce a variety of nAChRs with different functions and pharmacological properties.Previous studies have shown that nAChRs play important.roles in mediating the stimulation of tumor cell proliferation,migration,invasion and EMT in lung cancer.α5-nAChR is highly correlated with lung cancer progression and nicotine dependence.Recent studies have indicated that the CHRNA5 variant(encoding a5-nAChR)is strongly associated with lung cancer risk and nicotine dependence.Our previous studies have demonstrated that α5-nAChR mediates nicotine-induced lung cancer development and progression by activating various signaling pathways.These researches have indicated that a5-nAChR is an important therapeutic target for tobacco related lung cancer.In recent years,tumor-related inflammation has become a hot research,and inflammation is called the seventh characteristic of tumor.Mounting studies emphasize that chronic inflammation is crucial for the development of tumor.Inflammasome is a large cytosolic multi-protein complex that assembles in response to detection of infection-or stress-associated stimuli and results in the activation of inflammatory response.NLR family pyrin domain containing protein 3(NLRP3)inflammsome are well characterized and can be activated by many stimuli.NLRP3 inflammasome is involved in the development and progression of various tumors and has certain cell and tissue specificity.Increasing evidence has highlighted the importance of inflammation in lung cancer and a strong relationship between smoking and the NLRP3 inflammasome.However,it is unclear whether an inflammation-related effect of α5-nAChR contributes to cigarette smoking-related lung cancer.Here,we intended to investigate the association of α5-nAChR,STAT3,NLRP3 and clinical outcomes in LUAD,and the effect and mechanism of nicotine upregulation of NLRP3 signaling and promotes cell proliferation and migration in LUAD via the a5-nAChR/STAT3 axis.To our knowledge,this is the first study to show the association between a5-nAChR and the NLRP3 signaling axis in lung cancer,which may provide potential therapeutic targets for lung cancer treatments in the future.Objective1.Research the correlation of a5-nAChR and NLRP3 expression,smoking history,prognosis and other clinical characteristics in NSCLC samples.2.Investigate the role of a5-nAChR and NLRP3 inflammasome in NSCLC cell proliferation and migration in vitro.3.Determine the effects of a5-nAChR and NLRP3 inflammasome on tumorigenesis in NSCLC cell in vivo.Methods1.The correlation and prognosis of a5-nAChR,STAT3 and NLRP3 expression in NSCLC patients were analyzed by online database TIMER 2.0 and Kaplan-Meier Plotter.2.Immunohistochemistry(IHC)was performed to measure the correlation of a5-nAChR and NLRP3 expression,smoking history and other clinicopathological parameters in lung adenocarcinoma tissue microarray.3.Western blot was used to investigate the expression of a5-nAChR,p-STAT3,t-STAT3,NLRP3,pro-caspase-1 and caspase-1(p20)in A549 and H1975 cells treated with 1μM nicotine for 16 hours.4.The siRNA fragments of CHRNA5 were constructed and transfected into A549 and H1975 cell lines mediated by Lipofectamine 2000.We used western blot to detect the expression of a5-nAChR,p-STAT3,t-STAT3,NLRP3,pro-caspase-1 and caspase-1(p20)in control,si-NC and si-CHRNA5 group.5.A549 and H1975 cells were stimulated by nicotine and treated with si-CHRNA5 to detect the expression of IL-1β and IL-18 in cell culture supernatant.6.CHIP method was used to detect the presence of STAT3 binding site in NLRP3 promoter.7.The siRNA fragments of STAT3 were constructed and ransfected into A549 and H1975 cell lines with Lipofectamine 2000.Western blot was used to detect the expression of p-stat3,t-stat3,NLRP3,pro-caspase-1 and caspase-1(p20).8.Western blot was used to investigate the expression of NLRP3、pro-caspase-1、caspase-1(p20)in A549 and H1975 cells treated with LPS+ATP to activate NLRP3 inflammasome.9.CCK8 was performed to examine the effects of nicotine and LPS+ATP on the proliferation of lung cancer cells.10.Transwell assay and wounding healing assay were used to measure the effects of nicotine and LPS+ATP in lung adenocarcinoma cell migration.11.The CAM xenograft model was constructed.The chicken embryo was divided into control,nicotine,si-NC,si-CHRNA5,MCC950 and si-CHRNA5+MCC950 group.The lung tumor tissues of CAM were collected and embedded.HE-staining and immunohistochemical analysis were employed to detect α5-nAChR and NLRP3 expression in the above tissue sections.12.We detected α5-nAChR and NLRP3 expression by immunohistochemistry in mouse xenograft tissues.Results1.The results from the TIMER 2.0 online analysis tool showed that CHRNA5 and NLRP3 expressions were positively correlated with STAT3 expression,suggesting that CHRNA5 and NLRP3 may be positively correlated via STAT3 in the TCGA LUAD dataset.High CHRNA5 or NLRP3 expression correlated with poor prognosis,especially in LUAD,but not in LUSC by survival analysis using TCGA LUAD subset from the Kaplan-Meier plotter.2.We examined α5-nAChR and NLRP3 expression in 55 LUAD and 53 paracarcinoma tissue samples via IHC.The results showed that α5-nAChR and NLRP3 protein levels in LUAD tissues were significantly higher than those of paracarcinoma tissue samples.The expression of a5-nAChR correlated with NLRP3 using spearman’s correlation analysis in LUAD samples(P=0.018).In 25 LUAD specimens with smoking status,the expression ofα5-nAChR and NLRP3 in patients who smoked was higher than in samples of non-smoker.Expression of α5-nAChR and NLRP3 was correlated with smoking status(P=0.012;P=0.041)3.Western blot analysis showed that the expression of α5-nAChR,p-STAT3,NLRP3,pro-caspase-1 and caspase-1(p20)significantly increased by stimulation with nicotine compared to the control.No change in the expression of t-STAT3 was observed in any of the groups.4.The protein levels of α5-nAChR,p-STAT3,NLRP3,pro-caspase-1 and caspase-1(p20)decreased after siRNA-CHRNA5 treatment.5.Nicotine-induced extracellular levels of IL-1β and IL-18 significantly increased and the levels of IL-1β and IL-18 in the cell culture supernatants decreased after si-CHRNA5 treatment.6.Transcriptional element analysis showed that the NLRP3 promoter had a STAT3 binding sites.Therefore,a ChIP assays was performed to identify the binding of STAT3 to the NLRP3 promoter.The results showed that in A549 and H1975 cells,si-CHRNA5 treatment for 48h significantly reduced the binding amount of STAT3 and NLRP3 promoter compared with si-NC group.7.Western blot analysis showed that the protein levels of p-STAT3,t-STAT3,NLRP3,pro-caspase-1 and caspase-1(p20)were markedly decreased after siRNA-STAT3 treatment.8.The results showed that the expression of NLRP3,pro-caspase-1 and caspase-1(p20)was significantly increased by stimulation with 1 μg/mL LPS and 5 mM ATP in A549 and H1975 cells.9.The CCK-8 assay results demonstrated that the proliferation of lung cancer cells was significantly enhanced by nicotine or LPS/ATP alone and even stronger in combination.10.The results of the wound healing experiment indicated intensely faster wound closure by nicotine and LPS/ATP joint treatment than by nicotine or LPS/ATP alone in A549 and H1975 cells.The results of transwell assays ± Matrigel demonstrated that a nicotine and LPS/ATP combination increased A549 and H1975 cell migration and invasion compared to nicotine or LPS/ATP alone.11.We established A549 xenograft tumors in a CAM model.The nicotine-treated group promoted xenograft tumor growth compared with the control group.In addition,5 μM MCC950 sodium(a specific NLRP3 inflammasome inhibitor)or silencing of CHRNA5 suppressed xenograft tumor growth compared with the si-NC group.The si-CHRNA5+MCC950 group substantially inhibited tumor growth compared with the si-CHRNA5 group or MCC950 group alone,The tumor volumes of the nicotine-treated group were significantly larger than those of the control group.Conversely,the si-CHRNA5+MCC950 group was significantly smaller than those of the xenografts derived from si-CHRNA5 cells and A549 cells with MCC950.In the CAM model,α5-nAChR and NLRP3 expression was higher in nicotine-treated xenograft tissues than in the control group tissues.The expression ofα5-nAChR and NLRP3 was higher in the si-NC group than in the si-CHRNA5 tumor group or MCC950 tumor group.α5-nAChR and NLRP3 expression was significantly lower in the si-CHRNA5+MCC950 group than in the si-CHRNA5 or MCC950 group alone.12.α5-nAChR and NLRP3 expression was examined in mouse xenograft tissues.The expression of α5-nAChR and NLRP3 was higher in the NC(normal control)group than in the si-CHRNA5 tumor group.In nicotine-treated xenograft tissues,α5-nAChR and NLRP3 expression was higher than that in the NC group tissues.This result was also verified in the KD group and the nicotine-treated KD group.Conclusion1.Bioinformatics analysis shows that CHRNA5 and NLRP3 may be positively correlated via STAT3,and high CHRNA5 or NLRP3 expression correlated with poor prognosis in the TCGA LUAD dataset.2.The α5-nAChR was shown to correlate with NLRP3 in spearman’s interfix analysis in the LUAD specimens.Expression of a5-nAChR and NLRP3 was associated with smoking condition.3.Nicotine activates the NLRP3 inflammasome via α5-nAChR/STAT3 signaling in NSCLC cells.4.LPS/ATP activates the NLRP3 inflammasome and promotes nicotine induced NSCLC cell proliferation and migration.5.The functional effects of a5-nAchR and NLRP3 were confirmed in chicken embryo chorioallantoic membrane(CAM)and mouse xenograft models.Therefore,our findings uncover a new interaction between α5-nAChR and NLRP3,nicotine upregulates NLRP3 signaling and promotes cell proliferation and migration in LUAD via the α5-nAChR/STAT3 axis,which may provide potential therapeutic targets for lung cancer treat.