| At present,cancer is still the number one problem that plagues human health.Cancer research worldwide has gone deep into the level of targeted gene therapy.The incidence of prostate cancer(Prostate cancer,PCa)has been considered to be higher in Europe and the United States than in China,but with the improvement of people's living standards and the increased life expectancy,and serum prostate-specific antigen(Prostate specific antigene,With the increasing popularity of PSA screening,the incidence of PCa in China has also increased year by year.Since 2000,the incidence of prostate cancer in China has risen rapidly at a rate of 12.6%per year.Therefore,finding an effective method to prevent prostate cancer is a imperative issue.androgen deprivation therapy(ADT)is the first-line treatment of PCa,however,after more than ten months of ADT treatment,the majority of patients inevitably progress to castration-resistant prostate cancer(CRPC).This situation forces researchers to explore the molecular mechanism of PCa development from many angles and search for specific drugs and treatment methods.Recently,the scientists brought up cancer stem cells(CSC).CSCs are cells with self-renewal capacity and capable of producing heterogeneous cancer cells.CSCs account for a very small proportion of the tumors but have an important role in the survival,proliferation,metastasis and recurrence of the tumors.They have the potential of self-renewal and differentiation.Domestic researchers discovered a prostate cancer-specific stem cell(PrCSC),the percentage of which increases gradually during ADT treatment.PrCSC is believed to play a major role in prostate cancer progression and treatment resistance.The specific of PrSC is androgen independent besides general properties of CSC.Therefore PrCSC is more resistant to ADT treatment than other cancer cells.This study helps to screen potential targets for early identification of CRPC.More and more evidence shows that cancer stem cells are very likely to be the foundation of cancer recurrence,metastasis,and multidrug resistance.Therefore,screening cancer stem cells accurately,fully understanding their biological characteristics,and further specific drugs targeting cancer stem cells has become a research hotspot.CUL4B belongs to the Cullin family.It is an important components of Cullin-RING E3 Ubiquitin ligases(CRLs)and plays an important role in signal transduction,cycle regulation,and DNA damage repair.Our previous studies found that CUL4B is highly expressed in prostate cancer and promotes the invasion and metastasis of prostate cancer.We further found that CUL4B promoted cell proliferation of CRPC cells and not restricted to hormone stimulation.During hormone deprivation of LNCaP cells,the expression of CUL4B experienced a short-term decrease,and then gradually increased and tended to be stable.It is suggested that CUL4B has a certain correlation with the CRPC progression.Furthermore,CUL4B was found to promote the degradation of AR protein,and the regulation of CUL4B to downstream genes of AR pathway was unobvious.Therefore,we need to explore new mechanisms for CUL4B promoting CRPC progression.Bioinformatics analysis of expression microarray of LNCaP-NC/siCUL4B cells revealed that CUL4B was significantly associated with the development of prostate cancer stem cells.Through a series of CSC function experiments,it was found that CUL4B has the ability to promote tumor stem cells in vitro,suggesting that CUL4B may be key regulator of the prCSC.Next,by sending miR-seq microarrays,it was found that CUL4B can inhibit miR200b/c expression by histone epigenetic modification and therefore increase the expression of BMI1 protein,the downstream target molecule of miR200b/c,promoting PCa progression though maintaining the stem cell-like characteristics.In this study,a "CUL4B-miR200b/c-BMlI1" oncoprotein axial model was proposed in the progression of prostate cancer,linking CUL4B with non-coding RNA signaling pathways.It indicates that CUL4B plays an important role in the self-renewal and maintenance of prostate cancer stem cells,providing new insights and new targets for the clinical treatment of prostate cancer diseases from the perspective of stem cell therapy.Purposes:1.To explore the role of CUL4B in the progression of CRPC and its possible regulatory mechanisms2.Identification of CUL4B and CSC correlation though biological analyses of expression microarray and public datasets3.To test whether CUL4B molecules participate in the self-renewal and maintenance of prCSC4.Explore the molecular mechanism of CUL4B in promoting the regulation of prCSCMethods:1.Prediction of CUL4B associated phenotype by biological analyses of genome expression array of Lncap NC/siCUL4B cells1.I Detection of CUL4B protein expression in prostate cancer cell lines by western blot1.2 PCa cell line LNCaP was cultured by adherent cell culture technology.After transient transfection of siRNA targeting CUL4B,qPCR and western blot were used to detect the interference efficiency,and then the Lncap NC/siCUL4B cells were analyzed proceed human genome-wide expression profiling microarrays detection.1.3 Gene Set Enrichment Analysis(GSEA)was carried out to analyze the results of expression microarray profiling of Lncap NC/siCUL4B1.4 Analysis of CUL4B Expression in CRPC using Gene Expression Omnibus(GEO)Database2.Exploration of CUL4B in the progression of CRPC and possible mechanisms2.1 Detection of CUL4B expression in prostate cancer CRPC cells2.2 Identification of whether CUL4B expression is androgen responsive2.3 Expression of CUL4B during CRPC transformation2.4 Detection of CUL4B Regulation of AR and downstream genes of AR pathway3.Biological analysis of CUL4B in prostate cancer and stem cell correlationThe above-mentioned CUL4B-knocked genomic expression profiling chip was used to detect correlation with stem cells using GSEA analysis4.Testing whether CUL4B is involved in the self-renewal and maintenance of prostate cancer stem cells4.1 Cultured prostate cancer cells lines PC3 and DU 145 were transfected with lentivirus,and selected with puromycin for 2 weeks to construct stable CUL4B interference/overexpression PC3/DU145 cells.QPCR and western blot were used to detect CUL4B expression.4.2 The effects of CUL4B on the colony formation and self-renewal ability of prostate cancer cells detected by Clone Formation Assay in PC3 and DU 145 stable screening cells4.3 Sphere formation assay detects changes in the size and number of microspheres after PC3 cells stably interfere with CUL4B expression.4.4 Three-dimensional spheroid formation assay detects changes in the size of microspheres after PC3 cells stably interfere with CUL4B expression.4.5 Western Blot detection of CUL4B and prostate cancer stem cell-related markers c-Myc,Oct4,Sox-2,BMI1,CD44 expression correlation5.Molecular mechanism of CUL4B in the regulation of prostate cancer cell sternness5.1 Search for target genes downstream of CUL4B through GSEA analysis of gene expression profile,and verify theregulation in both mRNA and protein levels.5.2 Analysis of CUL4B and BMI1 correlation in Prostate adenocarcinoma(PRAD)dataset in The Cancer Genome Atlas(TCGA)database using MeV cluster software.5.3 To selected miRNAs that may be regulated by CUL4B,we integrated miRNA microarrays with target gene prediction software(miRWalk,miRanda,RNA22,and Targetscan)results and related literature reports,,and verified the regulation by qPCR.5.4 Chromatin immunoprecipitation(CHIP)assay to detect the enrichment of CUL4B in the promoter region of miR200b/cResult:1.CUL4B is associated with progression of CRPC in prostate cancer1.1 The expression of CUL4B in LNCaP cells is highest in prostate cancer cell lines,and the expression of CUL4B is higher in C42B and LNCaP-AI cells compared with LNCaP cells.1.2 GSEA with CUL4B knockdown genome expression profile indicated that CUL4B up-regulated genes were enriched in the CRPC phenotype(GSE77763)1.3 CUL4B expression in hormone-independent cells is higher than hormone-dependent cells in protein levels1.4 After androgen deprivation of LNCaP cells,qPCR detection of different concentrations/time DHT(Double Hydrogen Testosterone)stimulation,CUL4B expression did not change significantly1.5 Western Blot assay showed that the expression of CUL4B in LNCaP cells was decreased in a short time after hormone deprivation,then increased and maintained at a high level of expression.1.6 qPCR detection found that the regulation of CUL4B on PSA,TMPRESS2 and KLK4,downstream genes of AR pathway,was not obvious2.Analysis of correlation of CUL4B and prostate cancer sternness2.1 In the public databases GSE19713,genes up-regulated in CUL4B overexpression are enriched in stem cell-associated phenotypes.2.2 In vitro experiments confirmed that CUL4B molecules promote the regulation and maintenance of stemness in prostate cancer2.3 Cloning experiments confirmed that CUL4B promotes colony formation and self-renewal of PC3 and DU 145 cells.2.4 Serial sphere propagating assays revealed that CUL4B knockdown impaired the stem cell trait of self-renewal as assessed by secondary prostasphere establishment in PC3 cells.2.5 After CUL4B-knockdown PC3 cells were cultured in serum-free medium containing Matrigel for 14 days,the number of microspheres ultimately formed by a single cell was reduced compared to the control,the volume of microspheres formed at the same time was smaller,and the ability to dry out became weaker.2.6 CUL4B promotes the expression of c-Myc,Oct4,BMIl and CD44 in prostate cancer cells3.CUL4B inhibits the expression of miR200b/c by histone modification,and positively regulates BMIl,thereby promoting the regulation and maintenance of stem cells of prostate cancer.3.1 LNCaP cells transiently interfered with siRNA targeting CUL4B,and were submitted to the gene expression profile.Difference expressed genes were screened with a difference fold change>2 and P<0.05,and differentially expressed genes were analyzed by GSEA enrichment to find that the differentially expressed genes were enriched in BMI1,KRAS,RAPA and ATF2 signaling pathways,in which BMI1 molecules are related to stem cell progression.Verify that BMIl is positively correlated with CUL4B at the protein level,and this association was not found at the mRNA level.3.2 MeV software cluster analysis found a positive correlation between CUL4B and BMIl expression levels,P<0.00013.3 miRNA microarray analysis showed that 1144 miRNAs were up-regulated after CUL4B knockdown.We compared the 1144 miRNAs up-regulated with 4 target gene prediction software miRWalk,miRanda,RNA22,and Targetscan,combined with the proven control BMI1 in related literature.6 microRNAs were finally selected,miR128-3p,miR200b-3p,miR200c-3p,miR320a,miR183-5p,and miR218-5p,and then then miRNA were verified by qPCR.Among them,the fold change of miR200b/c was the most significant.3.4 CHIP experiments confirmed that CUL4B can directly bind to the promoter region of miR200b(S2,S3)and miR200c(S1),indicating the directly transcriptional regulation of CUL4B on miR200b/c.Conclusion:1.CUL4B promotes CRPC progression and functions without hormone stimulation2.CUL4B is associated with stem cell mantainess of prostate cancer cells3.CUL4B promotes self-renewal and maintenance of the properties of prCSC4.Progression and maintenance of stem cell-like characteristics of prostate cancer by CUL4B through the "CUL4B-miR200b/c-BMI1" regulatory axis... |
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