NCAPD3 Participates In The Molecular Mechanism That AR Regulates The Migration, Invasion And Cycle Transition Of Prostate Cancer Cells

Prostate cancer(PCa)is currently a leading cause of morbidity in the western male population.Androgen/androgen receptor(AR)signal pathway is necessary in the development of prostate cancer.Androgen/androgen receptor(AR)signal pathway drives growth of prostate cancer cells,and is closely involved in advanced prostate cancer metastasis.However,the molecular mechanisms of androgen-mediated cell metastasis are still not well-known.The condensin complex is essential for correct packaging and segregation of chromosomes during mitosis and meiosis in all eukaryotes.In vertebrates,there are two condensin complexes,condensin I complex and condensin ? 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.The functions of condensin ? complex during mitosis are well-established.Recently,additional roles for the human condensin II complex outside of mitosis have been characterized,including regulation of transcription.In Drosophila,NCAPD3 directly up-regulates genes required for innate immunity after systemic bacterial infection.It is also reported that NCAPD3 promotes bacterial clearance in human intestinal epithelial cells.However,the relationship between NCAPD3 and AR and the effect of NCAPD3 in prostate cancer cell migration and invasion induced by AR is still unknown.Early results(our unpublished results)revealed that androgen and androgen receptor(AR)can up regulate the expression of NCAPD3.Therefore we designed and finished the experiments as follows:Firstly,by using qRT-PCR and immunohistochemical analysis,we found that the mRNA and protein levels of NCAPD3 were significantly higher in prostate cancer tissue than that in normal prostate tissue.From RT-PCR and western-blot assays,we found that protein levels of NCAPD3 in androgen-dependent LNCaP and CWR22Rv1 cells were relatively higher than that in androgen-independent PC3 and DU 145 cells.The levels of NCAPD3 in normal prostate epithelial cell line WPMY-1 was the lowest.Thus,these results strongly suggested the high NCAPD3 levels was significantly correlated with prostate tumor in clinic,and the levels of NCAPD3 might be correlation with the progression of prostate cancer.Secondly,our results showed that Mib treatment significantly increaed NCAPD3 mRNA levels and protein levels after hormone-stripped for 3 days in LNCaP and CWR22Rv1 cell lines.Next,According to ChIP-seq results,we found three obvious AR binding peaks located within NCAPD3.Corresponding to CHIP-seq results,there were three AR binding sequences(ARBS)(named ARBS-1,ARBS-2 and ARBS-3).By using transcription factor binding sites prediction website JASPAR,we identified functional androgen responsive elements(ARE)in the three ARBS.Subsequently,by using dual-luciferase reporter assays and ChIP assay,we fully confirmed NCAPD3,a novel androgen-responsive gene,was up-regulated by androgen/AR axis in prostate cancer.Thirdly,LNCaP and CWR22Rvl cells were transfected with NCAPD3 siRNA and hormone-stripped for 48 hr immediately after transfection;and then cells were treated with 10 nM Mib for another 24 hr.From our results,Mib treatment significantly increased the protein levels of ETS-1,Snail and Cyclin D1,however knock-down of NCAPD3 resulted in a dramatic decrease in Mib-induced up-regulation effect.By performing wound healing assay,transwell migration and invasion assay,Mib-induced LNCaP and CWR22Rv1 cells migration and invasion were markedly impaired by NCAPD3 knockdown.Furthermore,flow cytometry analysis in LNCaP and CWR22Rv1 cells also implied that NCAPD3 played an important role in G1/S phase transition of cells induced by AR.Lastly,PC3 cells were transfected with expression vectors of AR and NCAPD3.LNCaP and CWR22Rv1 cells were transfected with siAR,siNCAPD3,or control siRNA,and cells were synchronized with nocodazole at mitotic.We found that the rod-shaped chromosomes were more clearly visible in the over-expressing AR and NCAPD3 cells compared with control cells.We also found that mitotic cells with abnormally condensed chromosomes were also observed in AR-depleted cells and NCAPD3-depleted cells.The chromosomes were often long and spindly and labeled only weakly or not in cells with antibodies of phospho-H3.However,chromosomes were condensed to rod-shaped and labeled strongly with an antibody of phosphorylated histone H3(phospho-H3)in control siRNA-transfected cells.These results thus suggest that the initial phase of mitotic chromosome condensation was induced,at least in part,by AR.In conclusion,NCAPD3 was a direct transcriptional target of AR in prostate cancer cells.NCAPD3 functioned as a positive regulator of cell cycle progression,cell migration and invasion.In addition,AR partly influence condensin ? complexes function.Taken together,our data suggest that NCAPD3 plays an important role in the progression of prostate cancer.