Chronic Stress Enforces Lung Adenocarcinoma Progression Via The ACh/α5-nAChR/FHIT Axis

ObjectiveLung cancer is one of the most common cancers worldwide and a leading cause of cancer-related death.Non-small cell lung cancer accounts for 80%-85%of all types of lung cancer,and lung adenocarcinoma is the main subtype.Smoking is a major cause of lung cancer,but adenocarcinoma of the lung often occurs in non-smoking patients and its cause is unknown.Chronic stress is closely related to the occurrence,development and prognosis of tumors,but the mechanism of chronic stress promoting lung adenocarcinoma remains unclear.Our previous studies have shown that the level of the parasympathetic neurotransmitter acetylcholine(ACh)increases and the expression of α5-nicotinic acetylcholine receptor(α5-nAChR)increases in the formation of lung adenocarcinoma grafts in mice induced by chronic stress.ACh mediated by α5-nAChR can promote the proliferation,migration and invasion of lung adenocarcinoma cells,suggesting that ACh/α5-nAChR axis-mediated chronic stress can promote the progression of lung adenocarcinoma.Our gene expression profile showed that α5-nAChR was positively or negatively correlated with DNA methyltransferase 1(DNMT1)and FHIT expression in lung adenocarcinoma.Therefore,the objective of this study was to investigate the promotion of chronic stress through ACh/α5-nAChR/FHIT axis in the progression of lung adenocarcinoma,and to provide a new strategy for the prevention and treatment of lung adenocarcinoma.Method1.Chronic unpredictable stimulation(CUMS)model and chronic restraint stress model(CRS)were established to stimulate subcutaneous tumor formation and/or tail vein metastasis in BALB/c nude mice by chronic stress.To investigate the role of ACh andα5-nAChR in the development and metastasis of lung cancer induced by chronic stress,and the expression and correlation of ACh,α5-nAChR,STAT3,DNMT1,FHIT,vimentin and MMP-9 in tumor tissues.2.The expression of α5-nAChR,STAT3,DNMT1,FHIT and their correlation with survival were analyzed by drawing survival curves of LinkedOmics online database.The correlation and clinical significance of α5-nAChR,DNMT1 and FHIT expression were analyzed by TIMER online database.3.The expression of α5-nAChR and FHIT in LUAD tissue microarray was detected by immunohistochemical staining.4.A549 and H1299 cells(con,CHRNA5+)were treated with different concentrations of ACh(1 μM,5 μM,10 μM,20 μM)for 48 h.The expression and correlation ofα5-nAChR,STAT3,DNMT1,FHIT,MMP-9 and vimentin were detected by Western blot.The effect of α5-nAChR expression level on ACh-induced expression of STAT3,DNMT1,FHIT,MMP-9 and vimentin were analyzed.5.A549 and H1299 cells(con,CHRNA5+)were treated with ACh for 48 h.EdU test,wound healing test and transwell migration and invasion assay were used to analyze the effect of α5-nAChR on the proliferation,migration and invasion of lung cancer cells.6.Chromatin immunocoprecipitation(ChIP)was used to detect the binding of STAT3 to DNMT1 promoter sites.7.The expression changes of α5-nAChR,DNMT1,FHIT,MMP-9 and vimentin in lung cancer cells induced by ACh were detected by STAT3 inhibitor.8.The expression of α5-nAChR,STAT3,FHIT,MMP-9 and vimentin were detected by DNMT1 inhibitor(5-AZ)in lung cancer cells,and the regulatory effect of DNMT1 on STAT3,FHIT,MMP-9 and vimentin was investigated.9.The role of FHIT methylation level in ACh-induced LUAD migration and metastasis was detected by MSP method.Results1.The tumor formation experiment of chronic restraint stress model showed that chronic restraint stress promoted the growth of transplanted tumor in mice and mediated the expression of α5-nAChR,FHIT,vimentin and MMP-9.2.The analysis results of database and clinical samples showed that CHRNA5 was correlated with the expression levels of STAT3,DNMT1 and FHIT in lung adenocarcinoma tissues and had clinical significance.CHRNA5 and FHIT were associated with poor prognosis and pathological stage in LUAD patients.3.Western blot results showed that the expressions of pSTAT3,DNMT1 and vimentin were increased and the expression of FHIT was decreased by α5-nAChR mediated ACh(p<0.05).4.EDU,Transwell and scratch experiments showed that ACh mediated the proliferation,migration and invasion of LUAD cells through α5-nAChR.5.ChIP assay showed that α5-nAChR mediated the binding of STAT3 to DNMT1 promoter in LUAD cells.6.Western blot results showed that vimentin expression of ACh/α5-nAChR was increased and FHIT expression was decreased through STAT3/DNMT1 mediation(p<0.05).7.MSP experiments showed that ACh/α5-nAChR mediated FHIT promoter methylation through STAT3/DNMT1.8.Chronic unpredictable stimulation(CUMS)tumor bearing mouse model showed that chronic stress mediated LUAD graft tumor growth and liver metastasis through ACh/α5-nAChR/DNMT 1/FHIT pathway.However,mecamylamine inhibited the tumor-promoting and metastasizing effects of this pathway.Conclusion1.In the progression of lung adenocarcinoma promoted by chronic stress,the expression levels of ACh and α5-nAChR were positively correlated.2.Chronic stress promotes the release of ACh,activation of α5-nAChR/STAT3/DNMT1 pathway mediates FHIT hypermethylation,and promotes the progression of lung adenocarcinoma.Related proteins may be potential targets for the future diagnosis and treatment of chronic stress-related LUAD.