| Objective:A body of preclinical evidence now demonstrates that estrogen is a driver of Non-Small Cell Cancer(NSCLC).This study was performed to elucidate the expression and functions of ERβ1,2 and 5,the major molecules of estrogen signaling in NSCLC tissues.Methods:Samples 113 patients with primary NSCLC(diagnosed between October 2008 and February 2014)who underwent surgery in the Department of Thoracic Surgery(affiliated to Tongji Hospital of Huazhong University of Science and Technology Tongji Medical College)were enrolled into the study.IHC staining was performed to detect nuclear and cytoplasmic staining of ERβ1,2,5,Insulin Growth Factor 1 Receptor(IGF1R)and Ki67.Association between expression of ERβ1,2,5 and IGF1R and Ki67,and clinicopathological characteristics include gender,age,smoking index,histological type,TNM stage,degree of differentiation and Overall Survival(OS)after surgery were analysed.The univariate survival analysis was performed using Kaplan-Meier method,survival curves were compared with the log-rank test.Additionally,univariate and multivariate COX regression analyses were carried out.Results:Overexpression of c/n ERβ1,2,5 was detected by immunohistochemical analysis in the NSCLC samples at different levels.High expression of Ki67 was detected in tissues with overexpression of cERβ1 and cERβ5.cERβ5 expression was significantly associated with elder than 55 years old patients,and cERβ1 strong expression(+++)was higher in IV stage patients.cER(31 expression was significantly associated with cER(35,IGF1R and Ki67 expression and cERβ5 xpression was associated with IGF1R and Ki67 expression.Strong expression of cERβ1 and cERp5was associated with worse overall survival.Multivariate analysis showed that cERβ1 and cERβ5 were independent prognostic markers of decreased overall survival.Conclusion:Overall,these findings support ERβ1 and ERβ5 play important roles in tumorigenesis and development of NSCLC.Potential accociation exists between estrogen signaling and IGF1 signaling.ERβ1 and ERβ5 may play different roles on the growth and progression of NSCLC and may be considered as a new prognosis predict marker and therapeutic target in NSCLC.Objective:The roles of ERβ3,ERβ2,ERβ5 in proliferation and metastasis of NSCLC are not completely appreciated in tumour development.And the relation between ERβ1 ERβ2,ERβ5 and IGF1 signal is still unknown.The aim of the present study is to determine the function and possible mechanism of ERβ3,ERβ2,ER(35 in NSCLC development.Methods:A549 and H1793 cells were transfected siRNA-ERβ1,siRNA-ERβ2,siRNA-ERβ5,pcDNA3.1-ERβ1,pcDNA3.1-ERβ2,pcDNA3.1-ERβ5,lentivirus-ERβ1,lentivirus-ERβ2,lentivirus-ERβ5.Immunofluorescence was used to detected location and stain of ERβ1,ERβ2,ERβ5 and IGF1R.Proliferation was analysed by CCK8 and Edu assays,invasion and migration was analysed by Transwell assays after transfection of siRNA-ERβ1,siRNA-ER(32,siRNA-ERβ5,pcDNA3.1-ERβ1 pcDNA3.1-ERβ2 and pcDNA3.1-ER(35.CCK8 and wound healing assays was to investigated growth and migration after transfection of pcDNA3.1-ERβ1 +siRNA-IGF1R,pcDNA3.1-ERβ2+siRNA-IGF1R,pcDNA3.1-ERβ5+siRNA-IGF1R.A549 cells transfected lentivirus-ERβ3,lentivirus-ERβ2,lentivirus-ERβ5,were subcutaneously injected in BALB/c mice to built xenograft tumor model.Tumor weight and volumes were calculated,and IGF1R expression in tumors were detected.Results:Green staining of ERβ3,ERβ2,ERβ5 located in both nucleus and cytoplasm,stain of ERP2,ERβ5 deepened after 17β-estradiol stimulation.Red staining of IGF1R mainly located in cytoplasm of A549 and H1793 cells.Administration with siRNA-ERβ1 and siRNA-ERβ5 in A549 and H1793 cells decreased cell growth,invasion and migration while pcDNA3.1-ERβ1 and pcDNA3.1-ERβ5 treatment accelerated proliferation and metastasis.However,treated with pcDNA3.1-ERβ1+siRNA-IGF1R,pcDNA3.1-ERβ2+siRNA-IGF1R,pcDNA3.1-ERβ5+siRNA-IGF1R,cell growth and migration were reversed in A549 and H1793 cells.In xenograft mice,gourp of lentivirus-ERβ1,lentivirus-ERβ5 demonstrated a dramatic decrease in tumour weight and volumes.In addition,transfection of lentivirus-ERβ1,lentivirus-ERβ2,lentivirus-ERβ5 weaken expression of IGF1R compared to control group.Conclusion:Therefore,our study strongly highlights the biological significance of the ERβ1 and ERβ5 identifies IGF1 as a key regulator in the NSCLC proliferation and metastasis.ERβ1 and ERβ5 promote proliferation and metastasis in NSCLC through IGF1 signal pathway.ERβ1 and ERβ5 may emerges as a new therapeutic target for Endocrine therapy of NSCLC.Further study will focus on the mechanism of IGF1 signal regulation by ERβ1 and ERβ5.Objective:Activation of ERβ1 and ERβ5 promote proliferation and metastasis of NSCLC through IGF1 signal pathway.While the exact mechanism of this phenomenon is still unknown.This study is aim to investigate the mechanism how ERβ1,ERβ2,ERβ5 regulate the IGF 1 signal.Methods:A549 and H1793 cells were transfected of siRNA-ERβ1,siRNA-ERβ2,siRNA-ERβ5.Total RNA was isolated from cells and real-time PCR was used to analysed relative expression of IGF1mRNA in each group.We identified six potential EREL(Estrogen Response Elements Like)sites in the promoter of IGF 1 gene,and chromatin immunoprecipitation analysis was applied to investigate if ERβ1 ERβ2,ERβ5 protein bind on the IGF1 promoter derectly.Dual luciferatse reporter analysis was used to detect if ERβ1,ERβ2,ERβ5 could activate IGF1 transcripts via these EREL sites respectively.Results:Relative expression of IGF1 mRNA decreased in siRNA-ERβ1 and siRNA-ERβ5 group in A549 and H1793 cells,with estradiol-dependent.Six potential EREL sites were predicted in the promoter of IGF1 gene,named site 1,ATGACATCATAACCC,site 2,AGGGGACAGTGACAG,site 3,ATGAGACAGTGCCCT,site 4,GTGTCTTCATGCCCA,site 5,TTGTCACCATGCCCA,site 6,TAACTTTGCCAG.Chromatin immunoprecipitation analysis found ERβ1,ERβ2,ERP5 did not bind to Non-ERE site.However,ERβ1 bind to site 2,site 5 and site 6,ERβ2 bind to site 1,site 3,site 4,site 5,and site 6,and ERβ5 bind to site 2,site 3 and site 4.Dual luciferatse reporter analysis showed ERβ1,2,5 activated transcription of IGF1 gene.The activated sites of ERELs is site 2,site 5 and site 6 to ERβ1,site 1,site 3 to ERβ2 and site 2,site 3 and site 4 to ERβ5,respectively.Conclusion:Our data highlight the importance of ERβ1,ERβ2,ER(35 in mediatingtranscription of IGF1 by binding to EREL sites respectively.Taken together,our studyindicating the exact mechanism that ERβ1,ERβ2,ERβ5 bind to IGF1 promoter with specific sites.Our study strongly highlights ERβ isoforms as new molecular classifications o f NSCLC from gender perspectives.ERβ isoforms could be new molecular classifications of diagnosis and new molecular classifications of prognosis predicters and therapeutic targets in NSCLC. |
PDF Full Text Request