NCAPD3 And CXCL13 Are Involved In AR-promoting PCa Xenograft Tumorigenesis And Preliminary Study Of NCAPD3 In PCSC

Prostate cancer(PCa)is the most common malignancy in the world with the morbidity and mortality second only to lung cancer,ranking second in the cancer mortality rate.Androgen/androgen receptor(AR)signal pathway is necessary in the development of prostate cancer.In vertebrates,there are two condensin complexes,condensin Ⅰ complex and condensin Ⅱ complex.NCAPD3 is a subunit of the condensin Ⅱ complex and present in nucleus.Previous study shows that NCAPD3 is an androgen-responsive gene in prostate cancer cells.Our previous experiments show that NCAPD3 is a target gene of AR and find that NCAPD3 plays an important role in androgen-mediated up-regulation of ETS-1,Snail and Cyclin D1,indicating that NCAPD3 is involved in the development of AR-promoted prostate cancer.Chemokines(’chemotactic cytokines’)are a family of lower molecular weight cytokines.Increasing evidence shows that chemokines play an important role in the survival,growth,migration,invasion,proliferation and apoptosis of cancer cells.In prostate cancer,serum levels of CXCL13 are positively correlated with prostate specific antigen(PSA).Our early experimental results show that androgen receptor(AR)up regulates CXCL13 expression and find that CXCL13 plays an important role in androgen-mediated up-regulation of ETS-1,Snail and CyclinB1.These results demonstrate,at the cellular level,that CXCL13 plays an important role in androgen-mediated prostate cancer cell proliferation.Next,we performed in vivo experiments in mice to verify whether NCAPD3 and CXCL13are equally involved in the development of AR-promoted prostate cancer cells in vivo.We constructed the cell lines stably expressing AR-N,NCAPD3 and CXCL13 by using the prostate cancer cell line CWR22Rv1.For NCAPD3 in vivo experiments,male nude mice were respectively divided into five groups for subcutaneous injections with control CWR22Rv1 cells,C WR22Rv1 cells overexpressing AR-N,CWR22Rv1 cells overexpressing NCAPD3,CWR22Rv1 cells knocking down NCAPD3(with siNCAPD3),CWR22Rv1 cells overexpressing AR together with knocking down NCAPD3.For CXCL13 in vivo experiments,male nude mice were respectively divided into four groups for subcutaneous injections with control CWR22Rv1 cells,CWR22Rv1 cells overexpressing AR-N,CWR22Rv1 cells overexpressing CXCL13,or CWR22Rv1 cells overexpressing AR-N together with knocking down CXCL13.The knock-down of NCAPD3/CXCL13 in vivo is performed via intratumoral injection with siRNA every 3 days for totally 3 times after the tumor was established to 50-100mm3.In the process,the tumor size and mice weight were measured at different days as indicated in figures.After tumors grown to 30 days,the differences of tumor growth among groups are analyzed by observing tumor morphology,measuring tumor size and weighting body weight of mice.We found that tumors that over-expressed AR-N,NCAPD3,and CXCL13 tumors were significantly larger than those of the control group,whereas tumors that knocked down NCAPD3 and CXCL13 were significantly less than those of the control group.Furthermore,western blot and immunohistochemistry are used to detect the expression of AR,NCAPD3,CXCL13,ETS-1,Cyclin D1,Cyclin B1 and Snail.All in vivo data here are consistent with the conclusion we obtained in vitro in prostate cancer cell lines,suggesting that NCAPD3 and CXCL13 are involved in AR-mediated regulations of cell growth and proliferation in PCa cells in vivo.Studies have shown that tumor stem cells play the key role in cancer cell growth,proliferation,metastasis and many other processes.Therefore,we explore the role of NCAPD3 in cancer stem cells and the specific molecular mechanism of promoting prostate cancer proliferation.Prostate cancer cell lines were cultured in medium containing B27 instead of serum to form tumor stem cell spheres.Western blot data showed that NCAPD3 had a low expression in tumor stem cell spheres,and the expression of AR in tumor stem cell spheres also showed low expression,and the effects of NCAPD3 on prostate cancer stem cells and specific molecular mechanisms still need to be further explored.