The Function And Mechanism Of α5-nAChR On EMT In NSCLC

BackgroundLung cancer is the second most prevalent malignant tumor in the world,and non-small cell lung cancer(NSCLC)is the most prevalent subtype,and due to its insidious nature and atypical initial symptoms,most patients have already developed distant metastasis by the time of diagnosis.metastasis in NSCLC is a pathological process with complex causative factors and difficult to control.NSCLC cells can acquire the ability to metastasize distantly by undergoing epithelial-mesenchymal transition(EMT),thus it is crucial to investigate the triggers and mechanisms of EMT development in lung cancer.Our previous study found that α5-nAChR and p-Stat3 activate each other and play an important role in nicotine promotion of NSCLC proliferation,but the role of α5-nAChR in EMT and metastasis of NSCLC cells remains to be investigated.Therefore,in the first part of our study,we investigated the hypothesis that α5-nAChR regulates the Stat3/Jabl signaling axis to achieve the regulation of EMT and distant metastasis in NSCLC cells.We found that Gli2 plays an important role in smoking-associated tumors,and we applied bioinformatics to analyze the effect of Glil/Gli2 on the prognosis of NSCLC patients,and found that only high expression of Gli2 predicted poor clinical prognosis and the presence of Gli2 binding sites in the α5-nAChR promoter region.Therefore,we investigated the hypothesis that "Gli2 regulates α5-nAChR to regulate EMT and metastasis of NSCLC cells" in the second part of the study.Objectives1.To study the function and mechanism of α5-nAChR regulating Stat3/Jab1 signaling pathway in EMT of NSCLC cells.2.To study the function and mechanism of Gli2 regulating α5-nAChR in EMT of NSCLC cells.Methods1.PartⅠ1.1 Online bioinformatics site to analyze the expression levels,correlation and impact on survival of α5-nAChR and Jabl in lung cancer.1.2 Immunohistochemical detection of α5-nAChR and Jabl expression levels in lung cancer tissue microarrays and analysis of their relationship with clinicopatholog-ical parameters.1.3 Western blot to detect the expression levels of α5-nAChR and Jab1 in A549 and H1299 under the effect of nicotine.1.4 Western blot assay:expression of EMT Marker after silencing Jab1;expression of overexpression of α5-nAChR,t-Stat3,p-Stat3,Jab1 and EMT Marker;expression of t-Stat3,pStat3 and EMT Marker after silencing CHRNA5;alone silencing of CHRNA5 or t-Stat3,compared with simultaneous silencing of CHRNA5+t-Stat3,the expression of Jab1.1.5 ChIP assay to detect the presence or absence of p-Stat3 binding sites in the Jab1 promoter region.1.6 Scratch and Transwell migration and invasion assays to detect:invasion and migration ability of A549 and H1299 cells after α5-nAChR overexpression;invasion and migration ability of A549 and H1299 cells after silencing α5-nAChR and Jab 1.1.7 Animal experiments:immunohistochemical methods to detect the expression levels ofα5-nAChR and Jab1 in subcutaneous tumor-forming tissues;HE staining to observe tumor lung metastasis.2.Part Ⅱ2.1 Bioinformatics website to analyze the effect of Gli2 expression level on survival.2.2 Immunohistochemical method to detect Gli2 expression level in lung cancer tissue microarray and analyze its relationship with clinicopathological parameters.2.3 Western blot method to detect the expression levels of Gli2 and α5-nAChR in A549 and H1299 under the effect of nicotine.2.4 ChIP assay to detect the presence or absence of Gli2 binding sites in the promoter region of α5-nAChR.2.5 Western blot method to detect the expression levels of α5-nAChR,Jabl and EMT marker after Gli2 overexpression and knockdown.2.6 Scratch and Transwell migration and invasion assays to detect the invasion and migration ability of A549 and H1299 cells after Gli2 overexpression and knockdown.2.7 Immunohistochemical methods were used to detect the expression levels of Gli2 andα5-nAChR in subcutaneous tumor-forming tissues;HE staining was performed to observe the tumor lung metastasis.Results1.PartⅠ1.1 α5-nAChR and Jab1 are highly expressed in NSCLC tissues and predict poor clinical prognosisBioinformatics analysis showed that both α5-nAChR and Jab1 were highly expressed in NSCLC and positively correlated.The survival of patients with high expression of α5-nAChR and Jab1 was lower than that of patients with low expression.Microarray immunohistochemical staining showed that α5-nAChR and Jab1 were highly expressed in cancer tissues.α5-nAChR and Jab1 expression was independent of gender and age(P>0.05)and positively correlated with TNM stage(P=0.013,P=0.036).Immunohistochemical results of subcutaneous tumor-forming tissues showed that α5-nAChR and Jab1 were highly expressed in the NC group compared with the KD group,and α5-nAChR and Jab1 were highly expressed in the A549 α5-nAChR+ group compared with the A549 Ctrl group.1.2 Nicotine stimulation increased the expression of α5-nAChR and Jab1 in NSCLC cell linesWestern blot results showed that α5-nAChR and Jab1 expression were positively correlated in A549 and H1299 cell lines.The expression of α5-nAChR and Jab1 was increased in a non-concentration-dependent manner using 1 μM and 10 μM nicotine acting on A549 and H1299 cells 16H.1.3 α5-nAChR regulates EMT and in vitro migration invasion of NSCLC cells through regulating Stat3/Jab1 pathwayWestern blot results showed that the expression levels of EMT marker N-cadherin and Vimentin decreased after silencing Jab1.t-Stat3 levels did not change significantly after α5-nAChR overexpression,and p-Stat3,Jab1,N-cadherin and Vimentin expression levels increased;a5 nAChR silencing resulted in no significant changes in t-Stat3 levels and decreased expression levels of p-Stat3,Jab1,N-cadherin and Vimentin;compared with silencing α5-nAChR or Stat3 alone,Jab1 expression levels were lowest after simultaneous silencing of α5-nAChR and Stat3.The results of ChIP experiments showed that p-Stat3 is a transcription factor of Jab1.The results of scratch and Transwell migration and invasion assays showed that the migratory invasion ability of A549 and H1299 cells was diminished after Jab1 silencing;the migratory invasion ability was enhanced after α5-nAChR overexpression;and diminished after silencing.1.4 α5-nAChR regulates the metastatic ability of NSCLC cells in vivoThe HE staining results of nude mice lung tissues showed that there were significantly more metastatic foci in the lungs of α5-nAChR+group than NC group.2.PartⅡ2.1 Gli2 is highly expressed in NSCLC tissues and predicts poor clinical prognosisBioinformatics analysis showed that patients with high Gli2 expression had lower survival than those with low expression.Immunohistochemical staining of microarrays showed that Gli2 was highly expressed in cancer tissues;Gli2 expression was independent of gender and age(P>0.05)and positively correlated with TNM stage(P=0.004).Immunohistochemical results of subcutaneous tumor-forming tissues showed that Gli2 and α5-nAChR were highly expressed in the Gli2+group compared with the Ctrl group.2.2 Nicotine stimulation increases the expression of Gli2 in NSCLC cell linesWestern blot results showed that the expression of Gli2 was most significantly increased when the duration of nicotine action was 12H or 16H and the action concentration was 2 μM or 10 μM.2.3 Gli2 regulates EMT and in vitro migration invasion of NSCLC cells through regulation of α5-nAChRThe expression levels of α5-nAChR,Jab1,N-cadherin and Vimentin increased after Gli2 overexpression;after knockdown,the expression levels of the above molecules decreased.The results of ChIP experiments showed that Gli2 is a transcription factor of a5nAChR.The results of scratch and Transwell migration and invasion assays showed that the migratory invasion ability of A549 and H1299 cells was enhanced after Gli2 overexpression;after silencing,their migratory invasion ability was diminished.2.4 Gli2 regulates the metastatic ability of NSCLC cells in vivoThe HE staining results of nude mice lung tissue showed that there were significantly more metastatic foci in the lungs of Gli2+group than NC group.Conclusions1.Gli2,α5-nAChR,and Jab1 are highly expressed in NSCLC tissues and predict poor clinical prognosis.2.Nicotine stimulation increases the expression of Gli2,α5-nAChR,and Jab1 in NSCLC cells.3.α5-nAChR promotes EMT and in vitro invasive migration of NSCLC cells through regulating Stat3/Jab1 signaling axis.4.Gli2 promotes EMT and in vitro invasive migration of NSCLC cells in vivo by regulating α5-nAChR.