Reciprocal Activation Of ?5-nAChR And STAT3 In Nicotine-induced Non-small Cell Lung Cancer Cell Proliferation

[Background]Lung cancer is the most frequent cancers in the world,the morbidity and mortality rate is the highest among the cancer.In particular,non-small cell lung cancer(NSCLC)accounts for 85%of all cases of lung cancer.Tobacco smoking is the major environment cause in lung cancer,and nicotine,the addictive component of cigarettes,induces lung cancer cell proliferation,invasion and migration via the activation of nicotinic acetylcholine receptors(nAChRs).Genome-wide association studies(GWAS)have identified that locus at chromosome 15q24-26 encode the a3,?4 and a5 subunits of nAChRs,which increase the risk of lung cancer development,especially CHRNA5 gene encoding a5-nAChR is especially relevant to lung cancer.However,the mechanism of this subunit in lung cancer is not clear.Nicotine binding to lung cancer cell membrane surface nAChRs,nAChRs channels cause conformational changes,opening the ion channel,and can activate multiple signaling pathways.Studies have confirmed that a5-nAChR can activate the PI3K/AKT and MAPK downstream signaling pathways for influence cell proliferation,invasion and migration.Bioinformatics analysis of the CHRNA5 promoter region found have possible STAT3 binding sites,but a5-nAChR and STAT3 interaction and its effect on lung cancer cell function remains to be investigated.In this paper,we intend to study the expression of a5-nAChR and STAT3 in non-small cell lung cancer and its relationship with clinical pathological parameters,also analysis the molecular mechanism of reciprocal activation of a5-nAChR and STAT3 in nicotine-induced non-small cell lung cancer cell proliferation,which is of great significance to clarify the role of a5-nAChR in the occurrence and development of NSCLC,and has important theoretical significance and practical application prospect for further study on the prevention and treatment of NSCLC.[Objective]1.Investigate the correlation of a5-nAChR with STAT3 expression,smoking history and other clinical pathological characteristics in lung adenocarcinoma tissue.2.Determine the mechanism between ?5-nAChR and STAT3 interactions and effects on nicotine promote the proliferation of lung cancer cells.[Methods]1.Immunohistochemistry was used to detect the expression level of a5-nAChR and STAT3 in lung adenocarcinoma.Analysis the correlation of a5-nAChR with smoking history and other clinical pathological characteristics in lung adenocarcinoma.2.Using database to analyze correlation of overall survival and a5-nAChR expression in adenocarcinoma who have smoking history,and the The Kaplan-Meier plotter was used to examine the prognosis of a5-nAChR and STAT3 co-expression in NSCLC patients.3.Western blot was used to measure the protein expression levels of a5-nAChR,JAK2/STAT3 under the stimulation of nicotine.4.siRNA fragment of CHRNA5 encoding a5-nAChR was constructed and transfected into lung cancer cell with lipofectamine 2000.After siRNA-CHRNA5 transfection,the expression of a5-nAChR,JAK3 and STAT3 in nicotine group,siRNA-CHRNA5 group and(nicotine+siRNA-CHRNA5group)was detected by RT-PCR and Western blot respectively.5.ChIP assay was used to detect whether CHRNA5 promoter has STAT3 binding site.6.siRNA-STAT3 fragment was constructed and then transfected into lung cancer cell with lipofectamine 2000,the expression of a5-nAChR,JAK3 and STAT3 in nicotine group,siRNA-CHRNA5 group and(nicotine+siRNA-CHRNA5group)was detected by RT-PCR and Western blot respectively.7.CCK-8 was performed to examine the effect of interfere ?5-nAChR and/ort STAT3 in nicotine-induced lung cancer cell proliferation.[Results]1.Immunohistochemical analysis were detected the expression of ?5-nAChR and pSTAT3 in 130 lung adenocarcinoma samples,78(60%)had overexpression of?5-nAChR and 87(66.9%)had activation of pSTAT3.Our analysis showed that of the 87 samples with overexpression pSTAT3,58 also had elevated ?5-nAChR levels(P=0.022).These data suggest that there is a significant relationship of co-expression of ?5-nAChR and pSTAT3.In addition,there were 25 cases of lung adenocarcinoma patients with a history of smoking,of which there were 19 cases of positive expression of ?5-nAChR,which showed that the expression of 5-nAChR was closely related to the history of smoking.2.The database result shows that high ?5-nAChR levels correlated with decreased survival probability who have smoking history.The Kaplan-Meier plotter shows that survival probability with ?5-nAChR(+)/STAT3(+)was much lower than that of groups with ?5-nAChR(-)/STAT3(+)(P=0.034)and ?5-nAChR(+)/STAT3(-)(P=0.026).3.Western blot showed that in A549,H1299 and H1975 NSCLC cell lines,the expression of ?5-nAChR,pSTAT3 were significantly increased at 1 ?M nicotine treatment compared with control group(P<0.05),no change was observed in the tSTAT3 expression4.siRNA-CHRNA5 group expression of ?5-nAChR,JAK2 and STAT3 were decreased compared with the si-NC group(P<0.05),the(1 uM nicotine +siRNA-CHRNA5)group expression of ?5-nAChR,JAK2 and STAT3 were decreased compared with the 1 ?M nicotine group(P<0.05).5.ChIP assay showed that STAT3 binding to the ?5-nAChR promoter.There is only low amount of pSTAT3 binds to the ?5-nAChR promoter in the lung cancer cells,stimulation with 1 ?M nicotine for 16h led to a marked increase in the pSTAT3 on the?5-nAChR promoter,transcription siRNA-STAT3 significantly decreased pSTAT3 binding to the ?5-nAChR promoter.6.siRNA-STAT3 group expression of ?5-nAChR,JAK2 and STAT3 were decreased compared with the si-NC group(P<0.05),the(1 uM nicotine +siRNA-STAT3)group expression of ?5-nAChR,JAK2 and STAT3 were decreased compared with the 1 uM nicotine group(P<0.05).7.The results of CCK-8 show that compare with the control group,1uM nicotine group significantly promoted the proliferation,cell proliferation was inhibited in the siRNA-CHRNA5 and siRNA-STAT3,when the cells were transfected with siRNA-CHRNA5 and siRNA-STAT3 combined,there is a significantly reduced cell proliferation compared to the control group.[Conclusions]1.?5-nAChR expression in lung adenocarcinoma tissue was significantlyhigher than corresponding adjacent paracancerous tissue,and there is a relationship between ?5-nAChR and smoking history.High a5-nAChR expression correlated with decreased survival probability who have smoking history,which means that a5-nAChR can be the potential therapy for lung cancer.2.Nicotine activated JAK2-STAT3 signaling pathway via a5-nAChR in lung cancer cells,pSTAT3 binded to the a5-nAChR promoter and activated the expression of a5-nAChR,which means that there is a feedback loop between ?5-nAChR and STAT3,which plays an important role in promoting nicotine-induced non-small cell lung cancer proliferation...