Molecular Mechanism Of NCAPD3 Regulating The Expression Of EZH2 And MALAT1 To Promote The Development Of Prostate Cancer

Prostate cancer is the second leading cause of cancer death among men in Western countries.The latest global cancer burden data show that prostate cancer has become the fourth most common and eighth most deadly disease in the world.With the increase of the elderly population and the change of living and eating habits,the incidence and mortality of prostate cancer in China are increasing year by year.However,the molecular mechanism of the occurrence and development of prostate cancer is still unclear,and further basic research is needed to improve it continuously,and provide a more solid theoretical basis for clinical treatment.NCAPD3 is one of the non-SMC regulatory subunits of Condensin II,which is mainly responsible for the condensation and segregation of chromosomes during mitosis.The previous research of our group found that NCAPD3 is highly expressed in prostate cancer tissues and cells,and promotes the occurrence and development of prostate cancer through EZH2.In addition,studies also found that LncRNA MALAT1 is a key RNA cofactor of EZH2 and promotes the function of EZH2 in CRPC.In this study,we employ prostate cancer cells,clinical tissues and model mice as experimental materials.IHC,Western blotting,qRT-PCR,ISH and other experimental methods are used to investigate the molecular mechanism of NCAPD3 regulating MALAT1 and EZH2 in prostate cancer and its important role in the occurrence and development of prostate cancer.Firstly,Western blot and qRT-PCR data show that NCAPD3,EZH2 and MALAT1 are all highly expressed in prostate cancer cells compared to normal and benign hyperplastic cells.Results of IHC and ISH also show that NCAPD3,EZH2 and MALAT1 are highly expressed in clinical tissues of prostate cancer.These results indicate that NCAPD3,EZH2 and MALAT1 are all highly expressed in prostate cancer tissues and cells,suggesting that the three have a certain correlation.Subsequently,after overexpressing or knocking down NCAPD3 in cells,the protein and m RNA levels of EZH2 increased or decreased accordingly.By jointly analysis of literature,transcriptome data and our experimental data,it is confirmed that transcription factors STAT3 and E2F1 involved in NCAPD3 regulating EZH2 gene expression.After overexpressing or knocking down NCAPD3,the levels of STAT3 and E2F1 increased or decreased accordingly.STAT3 or E2F1 inhibitors eliminate NCAPD3-induced upregulation of EZH2 in NCAPD3 stable overexpression cells.Nuclear and cytoplasmic separation experiments show that overexpression of NCAPD3 upregulates the levels of STAT3 and E2F1 in the nucleus.Co-IP experiments show that NCAPD3 interacts with STAT3 or E2F1,and CHIP experiments also confirm that overexpression of NCAPD3 increases the enrichment of STAT3 or E2F1 in the EZH2 promoter.These results suggest that NCAPD3 not only promotes the expressions of STAT3 and E2F1,but also recruits them to the promoter region of EZH2 to promote its gene expression.In addition,overexpressing or knocking down NCAPD3 in cells,the level of MALAT1 increased or decreased accordingly.It’s found that STAT3 mediates NCAPD3 regulating the gene expression of MALAT1.Treatment with Stattic,an inhibitor of STAT3,in NCAPD3 stable overexpression cells eliminates NCAPD3-induced upregulation of MALAT1.Overexpression of STAT3 in cells with NCAPD3 stable knocking down rescues the downregulation of MALAT1 induced by NCAPD3.CHIP experiment data show that overexpression of NCAPD3 increases the enrichment of STAT3 in the MALAT1 promoter.These results indicate that NCAPD3 promotes the transcription of MALAT1 by increasing the expression of STAT3 and recruiting more STAT3 to the MALAT1 promoter region.Finally,functional and in vivo experiments verify that NCAPD3 promotes the growth and migration of prostate cancer cells via the STAT3/E2F1-EZH2 and STAT3-MALAT1 pathway.After treating with Stattic,HLM006474(E2F1 inhibitor)or MALAT1-IN-1(MALAT1 inhibitor)in NCAPD3 stable overexpression cells,CCK-8,clone formation and wound healing experiments are carried out.These results show that NCAPD3 promotes the proliferation and migration of prostate cancer cells,and this promotion is weakened by the inhibitors.In vivo experiments of the xenograft tumor model in mice established with NCAPD3-stable overexpression PC-3cells and NCAPD3-stable knockdown CWR22Rv1 cells show that NCAPD3 enhances the growth of xenograft tumor;and the expressions of E2F1,STAT3,EZH2 and MALAT1 in xenograft tumor tissues are also regulated by NCAPD3.Taken together,this study confirms for the first time that NCAPD3 promotes the expressions of EZH2 and MALAT1 by increasing the levels of STAT3 and E2F1 and recruiting them to the EZH2 promoter or recruiting STAT3 to MALAT1 promoter,and further promoting the occurrence and development of prostate cancer.