The Clinical And Experimental Investigation Of KDM6B In Promoting Prostate Cancer Progression

Background:Histone methylation is an important field of epigenetics,which changes the steric configuration of DNA,leading to gene expression or repression.Histone methylation plays a vital role in the occurrence,development and recurrence of prostate cancer.H3K27 trimethylation(H3K27me3)is one of the most important repressive histone modification mediated by the histone methyltransferase enhancer of zeste homolog 2(EZH2).Methylated H3K27 contributes to the repression of various developmental genes transcription.Lysine(K)-specific demethylase 6B(KDM6B)could counter the effect of EZH2 and relieve the transcription repressive effects of H3K27me3,which can remove all three mono-,di-,or tri-methyl groups from methylated H3K27.It is involved in multiple cellular processes,including differentiation,proliferation,senescence,and apoptosis,which tend to effect responses,such as vertebrate development,cancer,inflammatory diseases,and neurodegenerative diseases and so on.Interestingly,KDM6 B expression could be induced by certain normal developmental cues and by stressful or pathogenic factors,such as inflammatory cytokines,cancerigenic factor,mitochondrial stress inducer and so on.Previous reports showed that abnormal expression or activity of KDM6 B was observed in malignant hematopoiesis,cervical carcinoma,pancreatic carcinoma,glioma,renal cancer and so on.But there is no research focusing on the expression,biological function and mechanism of KDM6 B in prostate cancer.Objectives:To clarify the expression profile of KDM6 B in prostate cancer and explore its value in prostate cancer prognosis assessment.Additionally,to investigate the biological function and molecular mechanism of KDM6 B in prostate cancer progression,and judge whether KDM6 B can be served as a target for the treatment of prostate cancer.Methods:Firstly,the research applied GEO(Gene Expression Omnibus)database and TCGA(The Cancer Genome Atlas)database to analyze the expression profile of KDM6 B mRNA in prostate cancer and explore its value in prognosis assessment.And immunohistochemistry was conducted in a self-constructed tissue microarray to explore KDM6 B protein expression profile and assess its prognostic value in protate cancer.In vitro,the PC3 and C4-2B cells with siRNA mediated KDM6 B deletion wereapplied to explore the effects of KDM6 B on proliferation,clone formation,migration,invasion,cell cycle and apoptosis in prostate cancer.For xenograft model,the in vivo-jetPEI Delivery Kit together with KDM6 B siRNA were used to knockdown the expression of KDM6 B by intratumor injection,and scramble siRNA was taken as the control.The tumor growth velocity and tumor volume were compared between groups.Additionally,KDM6 B inhibitor GSK-J4 was administrated to test its effects on clone formation,cell cycle and apoptosis in vitro and proliferation in vivo.Then,two GEO datasets,Dingtianlidi sequencing data and tissue microarray data were applied to assess the correlation between AR(androgen receptor)and KDM6 B.LNCaP cell was treated with DHT(double hydrogen testosterone)and MDV3100(enzalutamide)to observe the effects of AR activity on KDM6 B expression.ChIP-PCR was utilized to clarify whether DHT and MDV3100 administration affects the direct binding of AR to KDM6 B promoter in LNCaP cell.Further,a gene microarray was applied to compare the gene expression profile difference between wild type C4-2B cell and C4-2B cell with KDM6 B knockdown,and then screen out differentially expressed target gene.Then,two GEO datasets,Dingtianlidi sequencing data and tissue microarray data were applied to assess the correlation between target gene and KDM6 B.KDM6B knockdown and inhibition were conducted in PC3 cell to clarify whether these administrations could affect the target gene expression.ChIP-qPCR was utilized to clarify whether KDM6 B knockdown and inhibition could affect the abundance of H3K27me3 on the promoter of target gene in PC3 cell.Next,immnoprecipatation based mass spectmetry was conducted to search for a target transcriptional factor that binds to KDM6 B,and whether KDM6 B knockdown and inhibition could affect the binding of the target transcriptional factor to KDM6 B in PC3 cell was also investigated.Last,ChIP-qPCR was used to clarify whether KDM6 B knockdown and inhibition affects the binding of KDM6 B and target transcriptional factor to the promoter of the target gene in PC3 cell.Results:The mRNA sequencing data and tissue microarray data confirmed that KDM6 B was upregulated in prostate cancer,and even higher in metastastic prostate cancer and castration resistant prostate cancer.Besides,KDM6 B overexpression was observed in patients with high Gleason score.Survival analysis showed that KDM6 B could serve as an independent predictor for the early recurrence of prostate cancer.In vitro,siRNA mediated KDM6 B knockdown could surpress the proliferation,clone formation,migration and invasion of PC3 and C4-2B cell,increase sub-G0-G1 populations,decrease sub-S-G2 populations and induce more apoptosis in PC3 and C4-2B cells.In vivo,KDM6 B deletion in PC3 and C4-2B cell lines remarkably decreased the tumor volume and weight.Similarly,KDM6 B inhibitor GSK-J4 treatment inhibited the ability of PC3 and C4-2B cells to form colonies.Analysis of cell cycle distributions revealed increased sub-G0-G1 populations and decreased sub-S-G2 populations in PC3 and C4-2B cell lines treated with GSK-J4.Additionally,GSK-J4 also led to more apoptosis in PC3 and C4-2B cells.Further,administration of GSK-J4 suppressed the tumor growth velocity and volume in a C4-2B mice tumor model.A negative correlation between AR and KDM6 B was observed by analyzing mRNA sequencing data and tissue microarray data.It was indicated that DHT decreased the mRNA and protein expression of KDM6 B in LNCaP cell,while MDV3100 elevated the mRNA and protein expression of KDM6 B in LNCaP cell.Further,ChIP-qPCR showed that DHT increased AR binding to KDM6 B promotor which might then lead to KDM6 B expression suppression in LNCaP cell.In contrast,MDV3100 decreased these binding which might then lead to KDM6 B expression increase.Gene microarray analysis was conducted to compare gene expression profile between wild type C4-2B cell and C4-2B cell with KDM6 B knockdown and found reduced CCND1(cyclin D1)expression in C4-2B cell with KDM6 B knockdown.The mRNA sequencing data and tissue microarray data indicated a positive correlation between KDM6 B and CCND1 expression.qPCR and western blot indicated significantly reduced KDM6 B and CCND1 mRNA and protein expression in KDM6 B knockdown PC3 cells.Administration of GSK-J4 also decreased CCND1 mRNA and protein expression in PC3 cell.Then,ChIP-qPCR indicated decreased H3K27me3 abundance at KDM6 B promotor after the siRNA mediated KDM6 B knockdown and GSK-J4 mediated KDM6 B inhibition in PC3 cell,which then led to CCND1 expression suppression.To search for the transcription factor cooperating with KDM6 B,an immunoprecipitation targeting KDM6 B and subsequent mass spectrum were conducted.The mass spectrum identified smad2/3 directly or indirectly binding to KDM6 B.After the silencing and inhibition of KDM6 B in PC3 cells,the binding of smad2/3 to KDM6 B decreased.Next,ChIP-qPCR showed that siRNA mediated KDM6 B knockdown and GSK-J4 mediated inhibition of KDM6 B significantly decreased the binding of KDM6 B and smad2/3 to the promotor of CCND1 in PC3 cell.Conclusion:KDM6B was upregulated in prostate cancer,and even higher in metastastic prostate cancer and castration resistant prostate cancer.Besides,KDM6 B overexpression was observed in patients with high Gleason score.Additionally,KDM6 B could serve as a predictor for the early recurrence of prostate cancer.In vitro cellular biological researches and in vivo mouse model revealed the oncogenic role of KDM6 B in prostate cancer.Additionally,GSK-J4,as the inhibitor of KDM6 B,could suppress the vitality and progression of prostate cancer,and can be served as a promising agent for the treatment of prostate cancer.Furthermore,our study reveals the up-stream and down-stream mechanism of KDM6 B in prostate cancer.We first report that AR decreases the transcription of KDM6 B,and KDM6 B demethylates H3K27me3 on the promoter of CCND1 and cooperate with smad2/3 to prompt the expression of CCND1.