The Identification Of Smad4 Splicing Variants In Human Prostate And The Mechanism Of Their Function On Cell Proliferation And Apoptosis In Prostate Cancer Cells

Objective:Prostate cancer（PCa）is the secondary common cancer in the world,and its incidence in China is gradually increasing recently.Castration-resistant PCa which relapses within 12-18 months after surgical castration is more aggressive,but there has no effective clinical treatments on.The growth inhibition mediated by transforming growth factor beta（TGF-β）/Smads signal pathway in epithelial cells of normal prostate is lost in prostate cancer（PCa）.Smad4 is the key molecular in TGF-β/Smads signal pathway,and it was discovered to be null or mutated in many kinds of cancers but not in prostate cancer,indicating the different mechanism from the regulation of Smad4 with full length in PCa development.Based on the discovery of two additional Smad4 bands with smaller molecular weight in normal prostate tissue,compared to that in prostate cancer cells,this study is to identify the Smad4 spliced variants expressed in prostate,further to illustrate the impact of these spliced variants to proliferation,apoptosis of PCa cells and finally to explore the mechanism of Smad4spliced variants affecting the cell proliferation and apoptosis of PCa cells.Method:The DNA sequences of the two Smad4 variants are identified by RT-PCR,RACE and DNA sequencing together,and the domains of Smad4 variants are detected by Western blot using three Smad4antibodies against different domains of Smad4;Then three stable PCa cell lines PC3-empty vector,PC3-Smad4 V1 and PC3-Smad4 V2 are established by puromycin screening after lentivirus infections,and their proliferation,cell cycle and cell apoptosis are examined and compared separately by CCK-8,colony formation experiments and flow cytometry.Finally,the molecular mechanism of these two Smad4 splicing variants affecting the proliferation and apoptosis is explored by dual-luciferase reporter experiment,Western blot and chromatin immunoprecipitation.Results:In this study,it was found that the two Smad4 with smaller molecular weight expressed in normal prostate tissues are both Smad4splicing variants which lack partial intermediate amino acids,compared to Smad4 full length.And the proliferation and colony formation of Smad4V1 or V2 stable transfeted PC3 cells are significantly inhibited,whereas the apoptosis of them are increased slightly.In addition,Smad4 V1 stable transfeted cells arrest in G0/G1 phase and the percent of G2/M in Smad4V1 stable transfeted cells is decreased.Moreover,Smad4 V1 and Smad4V2 could also increase the expression of apoptotic promoting factor Bax and cell cycle inhibitor p21,while decrease the expression of cell cycle protein cyclin D1 and cell apoptosis inhibitor Bcl-2.Furthermore,both Smad4 V1 and Smad4 V2 can activate Smads signal pathway,and both directly bind or promote the binding of Smad4 full length to the SBE regions of p21^WAF1 promoter to increase the P21 expression.Conclusion:The two Smad4 with partial intermediate amino acids deletion can both directly activate and promote endogenous Smad4 full length-induced downstream transcription,to influence the expression of cell cycle related factors（such as p21 or Cyclin D1）and cell apoptosis related factors（such as Bcl-2 or Bax）,finally inhibit proliferation and promote apoptosis of PCa cells.