Clinical Significance And Underlying Mechanism Of Abnormal Expression Of BCL11A In TNBC

BackgroudGradually,the occurrence of cancer,in China and even the world,is becoming one of the most significant serious diseases threatening the health of human being.The statistics of China cancer data coming from CA Cancer J Clin 2015 demonstrats that,cancer is becoming the leading cause of death in China and is a major public health problem,with increasing morbidity and mortality.The data shows that breast cancer has the highest incidence in women.In China,the incidence of breast cancer accounts for nearly 15%of all other cancers in women,and the mortality occupies the first place.And the high-risk age of it is bewteen 30 to 59 years old.What’s more,with the steady and rapid rising of morbidity and mortality,breast cancer is threatening the life health of women and causing a serious social health problem.Triple-negative breast cancer(TNBC)is a special type of breast cancer,with some biological characteristics such as high aggressive,high recurrence rate and mortality,endocrine therapy invalidation and lacking of specific therapeutic targets.With these,triple-negative breast cancer is becoming a hotspot and difficulty in the field of breast cancer.Recently,some studies have discovered that the B-cell lymphoma/leukemia 11A(BCL11A)gene is overexpressed in triple-negative breast cancer and its exogenous overexpression promotes tumor formation.The study indicates that BCL11A is expected to be a new therapeutic targets of triple-negative breast cancer.Thus,in our study,we designed to investigate the molecular mechanism of BCL11A in the development and progression of triple-negative breast cancer,for the purpose to provide a new molecular target for the clinical diagnosis and treatment of triple-negative breast cancer.Objectives1.To investigate the expression of B-cell lymphoma/leukemia 11A in breast cancer,especially triple-negative breast cancer.2.To observe the impacts of B-cell lymphoma/leukemia 112A gene silence on the funcions of triple-negative breast cancer cell lines by shRNA.3.To study the correlation and interaction bewteen B-cell lymphoma/leukemia 11A and N-myc downstream regulated gene 1(NDRG1),β-catenin(β-cat),androgen receptor(AR).Methods1.Immunocytochemistry was used to detect the B-cell lymphoma/leukemia 11A protein expression and location in 140 breast cancer tissues microarray.2.The colony formation assay was used to measure the proliferating ability of MDA-MB-231and Hs 578T after B-cell lymphoma/leukemia 11A shRNA transfection.3.Cell migration and invasion ability of MDA-MB-231and Hs 578T was detected by Transwell and wound healing assay after B-cell lymphoma/leukemia 11A shRNA transfection.4.The change of cell cycle in MDA-MB-231and Hs 578T was observed by flow cytometry after B-cell lymphoma/leukemia 11A shRNA transfection.5.B-cell lymphoma/leukemia 11A shRNA stably transfected into triple-negative breast cancer cell lines,MDA-MB-231and Hs 578T,Western blot assay was used to detect the protein expression of B-cell lymphoma/leukemia 11 A,N-myc downstream regulated gene 1,β-catenin,and androgen receptor.6.Western blot assay was used to observe the protein expression of androgen receptor and B-cell lymphoma/leukemia 11A after the treatment of androgen receptor agonist dihydrotestosterone(DHT)and androgen receptor antagonist bicalutamide.Results1.Of the 140 tumor tissues microarray,the B-cell lymphoma/leukemia 11A was overexpressed in triple-negative breast cancer,distributing in the cytoplasm and nucleus mainly.The expression of BCL11A has positively related with estrogen receptor(ER)status(χ2=80.545,P=0.000),progesterone receptor(PR)status(χ2=62.177,P=0.000),human epithelial growth factor receptor 2(HER2)status(χ2=9.503,P=0.009)and AR status(χ2=4.223,P=0.040),and does not have significantly connection with age(χ2=0.946,P=0.331),histological(χ2=3.278,P=0.166),and TNM stage(χ2=0.714,P=0.700).2.Univariate Cox proportional hazards regression model shows that TNM stage(P=0.004),ER status(P=0.044),PR status(P=0.043)and BCL11A status(P=0.014)have significant effects on the overall survival(OS),and age(P=0.272),tumor size(P=0.419;P=0.126),number of lymph node metastasis(P=0.994;P=0.015;P=0.174),histological(P=0.928),HER2 status(P=0.634)and AR status(P=0.404)do not have any effect on the OS.Multivariate Cox proportional hazards regression model shows that TNM stage(P=0.002,HR=2.543)and BCL11A status(P=0.020,HR=2.099)are the independent prognosis factors for the overall survival of breast cancer.3.Kaplan-Meier analysis shows that high BCL11A expression was associated with poor OS(χ2=6.367,P=0.012)in breast cancer.4.In the analysis of correlation,we found that there is a significant positive correlation bewteen BCL11A expression and AR stasus(r=0.174,P=0.040).5.We have found that BCL11A can promote the proliferation of triple-negative.breast cancer cell lines through the colony formation assay.Using transwell and wound healing assay to explore the role of BCL11A in the cell migration and cell invasion of breast cancer,we also have found that BCL11A interference suppressed cell migration(P<0.001;P<0.001)and cell invasion(P<0.001;P<0.001).Last,the results of flow cytometry demonstrated that BCL11A has no influence on cell cycle of triple-negative breast cancer cell lines.6.With the BCL11A shRNA transfecting to triple-negative breast cancer cell lines,MDA-MB-231 and Hs 578T,we found that the expression of NDRG1 in protein levels was increased,and the expression of BCL11A,AR and β-catenin in protein levels were reduced.7.After the treatment of DHT,the expression of AR in protein levels was increased,and the expression of BCL11A in protein levels was no change in MDA-MB-231 and Hs 578T.In addition,after the treatment of bicalutamide,the expression of AR in protein levels was reduced,and the expression of BCL11A in protein levels was no change too.