AR And Sp1 Co-regulate EphA3 In Prostate Cancer Cells

Prostate cancer is a common disease in men,and the incidence of prostate cancer is the second most lethal cancer in Western countries.The incidence of prostate cancer in China has increased year by year.So far,there is still no better cure for advanced prostate cancer.To treat early onset prostate cancer,androgen deprivation therapy has a therapeutic effect.When prostate cancer is transformed into androgen-independent prostate cancer,there is no way to cure it clinically.AR plays an important role in the progression of androgen-dependent prostate cancer developed to androgen-independent prostate cancer.Thus,the research for prostate cancer could not stripe AR.AR has been changed in the progression of prostate cancer,but the role of AR is always being present.Therefore,the study of AR signaling pathway is getting a breakthrough in the treatment of prostate cancer.In order to understand the progression of prostate cancer and to find a breakthrough for treatment,we should find other downstream genes of AR.EphA3 belongs to receptor tyrosine kinases family of Eph receptor subfamily.And it is the first cloned molecule in the Eph family.Recent studies show that,EphA3 has been upregulated in many cancers,and its mutations also occur in some cancers.However,in prostate cancer,the research of EphA3 is little done.By the early exploration,we found that the AR can affect the expression of EphA3.To address this problem we have designed the experiments as follows:First,we examined AR and EphA3 in the WPMY-1,LNCaP and 22Rv1 cells taking advantage of DHT stimulation,overexpression of AR,suppression of AR and other means of dealing with LNCaP and 22Rv1 cells.With the stimulation of DHT the expression of EphA3 is upregulated.Overexpressing AR in 22Rv1 cells was also found to increase the expression of EphA3.After suppressing AR by siRNA in 22Rv1 cells,we found the expression of EphA3 rendered down.So we suppose that AR can affect the expression of EphA3.Subsequently,we constructed EphA3-Luc(-789),EphA3-Luc(-317),EphA3-Luc(-237),EphA3-Luc(-789mSpl)and other EphA3 promoters for luciferase reporter gene assay to study promoter activity of EphA3.It was found that the activity EphA3-Luc(-789)and EphA3-Luc(-317)was raised in the case of overexpression of AR,but decreased in the case of the inhibition of AR,indicating that AR can modulate the expression of EphA3 promoter.The activity of EphA3-Luc(-237)was not changed when AR rose or decreased,indicating that Sp1 binding sites for EphA3 promoter expression plays a vital role.And the activity of EphA3-Luc(-789 mSp1)was also not changed when AR rose or decreased,indicating that AR function and Sp1 binding site have an important relationship.Therefore,we conclude that AR affects the expression of EphA3 promoter by affecting SP1 binding site.Next,in the 22Rv1 cells,we used mithramycin and siSp1 to inhibit Sp1,and found no significant change in the expression of Sp1,but EphA3 expression was down-regulated.The results further validate the luciferase reporter gene assay.Finally,we used chromatin immunoprecipitation experiments and protein immunoprecipitation experiments,and found that AR and Sp1 can combine to the promoter(-246～-243)of EphA3,Sp1 and AR can form a complex on LNCaP and 22Rv1 cells.So,we think,AR first combines with Sp1.Then the complex of AR and Sp1 binds to EphA3 promoter site(-246～-243).In summary,this study confirms that the complex of AR and Spl jointly regulates the expression of EphA3.