Nicotine May Promote Tongue Squamous Cell Carcinoma Progression Through Wnt/?-Catenin And Wnt/PCP Pathways

Tongue squamous cell carcinoma(TSCC)is one of the most common cancers in the neck and head.TSCC will seriously affect the patient's chewing,language and appearance functions,and after surgery and radiotherapy,five-year survival rate of TSCC patients is still only 50-70%.Studies have shown that smoking is one of the important risk factors for tongue cancer,and tobacco abuse is associated with a high incidence of TSCC.However,the specific mechanism of smoking caused by tongue cancer is still unclear.Studies have shown that nicotine in tobacco can promote lung cancer and esophageal cancer cell proliferation and invasion.There is little research on the effects of nicotine on TSCC.Wnt signaling pathway has been shown to be associated with the development,progression and metastasis of various tumors.Studies have shown that in tongue cancer some key proteins of Wnt pathway are aberrantly expressed,which are closely related to the survival rate of patients,metastasis and recurrence.However,whether smoking is related to the abnormal activation of the Wnt signaling pathway in TSCC has not been studied.The purpose of this study is as follows:In 33 cases of tongue squamous cell carcinoma(TSCC),to study the correlation between smoking and key proteins of Wnt signaling.To study the effects of nicotine on the proliferation and invasion of tongue squamous cell carcinoma cell line Cal27 in vitro and its preliminary mechanism.Part one,the relationship between smoking and the expression of key proteins in Wnt signaling pathwayObjective:To investigate the correlation between smoking and Wnt protein expression(Wnt5?,Ror2,?-catenin,c-jun)in 33 cases of tongue squamous cell carcinoma(TSCC).Methods:Immunohistochemical technique was used to detect the expression of Wnt signal protein in 33 cases of tongue squamous cell carcinoma(TSCC).Results:IHC staining showed that Wnt5a(IRS = 5.58 ± 2.32)and Ror2(IRS =6.05 ± 2.13)were significantly higher in non-smokers(IRS = 3.53±2.17)and(IRS =4.33 ± 2.13)(P= 0.013 and p = 0.019).In the smoking group,89.47%of patients had abnormal expression of ?-catenin,but only 53.33%(p = 0.018)in non-smokers.The c-jun expression in non-smoking group and smoking group is(IRS = 4.53±2.01 vs.IRS = 4.00 ±1.89,p = 0.442)separately.Conclusion:The expression of ?-catenin,Wnt5 and Ror2 in TSCC may be related to smoking.Part two,the nicotine-induced tongue squamous cell line Cal27 migration,invasion and Wnt pathway activationObjective:To investigate the effects of nicotine on the proliferation,migration and invasion of Cal27 in vitro and Wnt pathway.Methods:The control group(DMSO group),low concentration group(0.1 uM),medium concentration group(1 uM)and high concentration group(10 uM)were used.(1)The expression of ?-catenin,c-Myc,E-cadherin,p-GSK3b,Ror2,Wnt5a and c-jun were detected by Western blot in Cal27.2)TOP/FOP reporter system was used to detect the transcriptional activity of ?-catenin downstream transcription factor TCF;(3)Immunofluorescence staining(IF)was used to detect changes in ?-catenin at the cellular level;(4)The effect of nicotine on the proliferation of tongue squamous cell carcinoma cell lines was examined by CCK8 cell proliferation assay.(5)The effect of nicotine on the migration of tongue squamous cell carcinoma cell lines was examined by Wound healing assay.(6)Transwell assay was used to detect the effect of nicotine on the invasion of tongue squamous cell carcinoma cell lines.(7)To determine whether the effect of nicotine on Cal27 and on the Wnt signaling pathway is through?7 nAChR,a nicotine receptor inhibitor(a-BTX)was used.Results:Nicotine can activate Wnt/?-catenin and Wnt/PCP signaling pathway in vitro.Nicotine can significantly increase the proliferation,invasion and migration of Cal27.The above effects of nicotine is through ?7 nAChR.Conclusion:Nicotine can activate Wnt/?-catenin and Wnt/PCP signaling pathway through nicotine receptor and accelerate the proliferation,migration and invasion of tongue squamous cell carcinoma cell line Cal27.