| 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.Smoking is still the most important risk factor for lung cancer.Nicotine promotes the proliferation,migration,invasion,epithelial-mesenchymal transition(EMT)and inhibits cell apoptosis of lung cancer.Cell migration plays an important role in the process of tumor metastasis.Therefore,elucidating the molecular mechanism of cell migration is of great significance to prevent the metastasis and development of lung cancer.Nicotinic acetylcholine receptor(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(encodingα5-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 α5-nAChR is an important therapeutic target for tobacco related lung cancer.While the role of α5-nAChR in lung cancer cellular migration remains to be clarified.Lymphocyte antigen 6(Ly6)proteins are important diagnostic tools or endogenous regulators.The combination of Ly6 and nAChRs is involved in disease progression,and Ly6 can be a basis for the design of new drugs.Ly6E,a glycosylphosphatidylinositol(GPI)anchored cell surface protein,is another member of the Ly6 gene family.Previous evidence has shown that Ly6E is involved in human malignancies and is the potential therapeutic target for cancer immunotherapy.The expression of the Ly6E gene(LY6E)in lung,breast,gastric,colorectal,esophageal,head and neck and pancreatic cancer is higher than that in adjacent normal tissues.Ly6E promotes cancer progression,immune escape and drug resistance through TGF-β signaling.The TGF-β-mediated EMT is characterized by the molecular alteration of EMT markers such as E-cadherin,N-cadherin,Zebl and vimentin,followed by morphological changes that enable cell migration.In this paper,we intend to study α5-nAChR,Ly6E expression and clinical outcome of lung adenocarcinoma,also analysis the mechanism of a5-nAChR correlates with Ly6E and regulates NSCLC migration via TGF-β/Smad signaling in NSCLC,which is of great significance for studying the therapeutic targets of smoking-related non-small cell lung cancer.Objective1.Research the correlation of α5-nAChR and Ly6E expression,smoking history,prognosis and other clinical characteristics in NSCLC samples.2.Investigate the role of a5-nAChR and Ly6E in NSCLC cell migration in vitro.3.Determine the effects of a5-nAChR and Ly6E on tumorigenesis and metastasis in NSCLC cell migration in vivo.Methods1.The expression of CHRNA5 and LY6E in an array of human tumors was compared to the relative normal tissue using the GEPIA2 and UALCAN database.The overall survival(OS)analysis of CHRNA5 and LY6E was obtained using Kaplan-Meier Plotter.To test the prognostic value of the high coexpression of CHRNA5 and LY6E in lung adenocarcinoma,we downloaded a5-nAChR and Ly6E gene expression data from UCSC’s Xena cancer genomic browser.2.Immunohistochemistry(IHC)was performed to measure the correlation of α5-nAChR and Ly6E expression,smoking history and survival time of patientsmin lung adenocarcinoma tissue microarray.3.Western blot was used to investigate the expression of a5-nAChR,Ly6E,pSmad3,Smad2/3,vimentin,N-cadherin and Zebl in A549 and H1 975 cells treated with 1μM nicotine for 16 hours.4.The siRNA fragments of CHRNA5 and LY6E were constructed and transfected into A549 and H1975 cell lines mediated by Lipofectamine 2000.Western blot was used to detect the expression of a5-nAChR,Ly6E,pSmad3,Smad2/3,vimentin,N-cadherin and Zeb1 in si-NC,si-CHRNA5,si-LY6E and si-CHRNA5+si-LY6E group.5.In the above cell models,transwell assay and wounding healing assay were used to measure the effects of α5-nAChR and Ly6E in lung adenocarcinoma cell migration.6.The CAM xenograft model was constructed.The chicken embryo was divided into si-NC,si-CHRNA5,si-LY6E and si-CHRNA5+si-LY6E group.The lung tumor tissues of CAM were collected and embedded.HE-staining and immunohistochemistry were used to detect the expression of a5-nAChR,Ly6E,vimentin and Zebl in the above tissue sections.Results1.Bioinformatics online database indicated that CHRNA5 and LY6E were overexpressed in lung adenocarcinoma.High CHRNA5 and LY6E expressions significantly correlated with decreased survival probability in the TCGA lung adenocarcinoma subset.The survival time was lower for patients with high CHRNA5 and LY6E coexpression than for those with high expression of CHRNA5 or LY6E alone in lung adenocarcinoma.2.α5-nAChR and Ly6E expression was examined via immunohistochemistry in tissue microarrays containing 55 NSCLC and 55 paracarcinoma tissue samples.The results demonstrated that a5-nAChR and Ly6E protein levels in lung adenocarcinoma tissues were significantly higher than those in adjacent noncancerous tissues.Spearman’s correlation analysis showed a correlation between a5-nAChR and Ly6E expression in lung adenocarcinoma.In 25 adenocarcinoma specimens from smokers,23 cases of a5-nAChR expression(P=0.000),22 cases of Ly6E expression(P=0.002)were positive.These results indicated that the expression of a5-nAChR and Ly6E was correlated with smoking status.3.Western blot demonstrated that α5-nAChR,Ly6E,pSmad3,Zebl,N-cadherin and vimentin expression increased in the nicotine treatment group compared to the negative control(NC)group.No change in the expression of total Smad2/3 was observed in any of the groups.4.Compared with si-NC,silencing α5-nAChR downregulated a5-nAChR,Ly6E,pSmad3,Zebl,N-cadherin and vimentin expressions.Compared to si-CHRNA5 cells or si-LY6E cells alone,the expression of pSmad3,Zebl,N-cadherin and vimentin was significantly lower in si-CHRNA5+si-LY6E cells.5.Transwell assay and wound healing assay results had shown that the downregulation of α5-nAChR or Ly6E decreased cell migration compared to the si-NC group.Cell migration was significantly reduced in the si-CHRNA5+si-LY6E group compared to the si-CHRNA5 group or si-LY6E group alone.6.The CAM xenograft model had confirmed that the silencing of CHRNA5 or LY6E suppressed xenograft tumor growth compared with the si-NC group.Tumor volumes of si-CHRNA5+si-LY6E group were significantly smaller than those of the xenografts derived from si-CHRNA5 cells or si-LY6E cells.In the CAM model,the expression of a5-nAChR,Ly6E,Zeb1 and vimentin was higher in the si-NC group than in the si-CHRNA5 tumor group or in the si-LY6E tumor group.α5-nAChR,Ly6E,Zebl and vimentin expression was significantly lower in the si-CHRNA5+si-LY6E group than the si-CHRNA5 or si-LY6E group alone.Conclusion1.Bioinformatics analysis shows that CHRNA5 and LY6E expression increased and the overexpression was associated with poor prognosis in lung adenocarcinoma patients.2.The expression of a5-nAchR was associated with Ly6E,smoking status and decreased survival probability in non-small lung cancer.3.In vitro,α5-nAChR mediated Ly6E,the phosphorylation of the TGF-β1 downstream molecule Smad3(pSmad3,a key mediator of TGF-β1 signaling),the EMT markers such as Zeb1,N-cadherin and vimentin expression in NSCLC cells.The downregulation of Ly6E reduced α5-nAChR,pSmad3,Zebl,N-cadherin and vimentin expression.Functionally,silencing both a5-nAChR and Ly6E significantly inhibited cell migration compared to silencing a5-nAChR or Ly6E alone.4.The functional effects of a5-nAchR and Ly6E were confirmed in chicken embryo chorioallantoic membrane(CAM)and mouse xenograft models.Therefore,our findings uncover a new interaction between a5-nAChR and Ly6E that inhibits cancer cell migration by modulating the TGF-β1/Smad signaling pathway in NSCLC,which may serve as a novel target for therapeutic intervention. |
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